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High pressure and microwave assisted generation and pyrolysis-GC/MS analysis of glycated proteins

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HIGH PRESSURE AND MICROWAVE ASSISTED
GENERATION AND PYROLYSIS-GC/MS ANALYSIS
O F GLYCATED PROTEINS
By
Pik Kei LI
Department o f Food Science and A g r ic u ltu r a l Chemistry
M acdonald Campus, M cG ill University
Montreal, Quebec
A thesis submitted to the Faculty of Graduate Studies and Research in partial
fulfillment for the r e q u ir e m e n ts for the degree o f M aster o f Science.
August, 2002
® Peggy Li
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D E D IC A T IO N
This thesis is dedicated to m y m other (W e n d y ), m y father (David)
and my sister (Margaret).
II
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ABSTRA CT
The extent o f denaturation and glycation o f lysozyme and BSA with the
application o f high hydrostatic pressure (HHP) at 400 M Pa at 30°C from 8 to 48 hours
and focused m icrowave irradiation at 50°C under varying m icrowave power and from 10
to 60 m inutes was investigated in the presence and absence o f D-glucose. The HHP
treatm ent caused 10 to 20% denaturation o f lysozyme whereas m icrowave irradiation
caused 20 to 40% denaturation, with more destruction to the lysozyme in the presence o f
glucose com pared to the control. The extent o f glycation was also higher with the high
pressure samples, causing 60% glycation upon 8 hours o f high pressure exposure, but
decreasing to around 40% thereafter. M icrowave irradiation brought about 40% glycation
to the lysozym e samples upon 20 m in o f irradiation. BSA, on the other hand, was more
susceptible to damage by high energy exposures. BSA samples were denatured to a
greater extent compared to lysozyme, up to 80% upon the prolonged exposures, but in all
treatments, glucose seemed to act as a protectant contrary to the case o f lysozyme. The
extent of glycation detected was also minimal, ranging from 8 to 20%.
The feasibility o f analyzing glycated proteins using pyrolysis-GC/M S was also
investigated. Taking advantage o f the form ation o f a diagnostic m arker - 2,3-dihydro3,5-dihydroxy-6-m ethyl-4H-pyran-4-one - upon pyrolysis o f glycated proteins, the
intensity o f this peak was used to correlate the extent o f glycation. The intensity o f this
peak in the pyrogram s o f glycated lysozymes was found to increase linearly with
increasing incubation times and subsequently with the sugar loads o f the glycated
lysozyme. In addition, using the pyrogram s as unique fingerprints, the extent o f structural
changes between m odified and unm odified proteins were also assessed.
Ill
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RESUM E
L ’etendue de la denaturation et de ia glycosylation du lysozyme et du BSA sous
1’effet d ’une pression hydrostatique elevee (High Hydrostatic Pressure (HHP)) de 400
M Pa a 30°C pour des temps variant entre 8 et 48 heures, de m em e que I’irradiation sous
champ m icro-ondes focuse a 50°€ sous puissance variable et a des temps variant entre 10
et 60 m inutes, fut examinee avec et sans D-glucose. Le traitement HHP donne un taux de
denaturation du lysozyme de 10 a 20% , tandis que I5irradiation micro-ondes donne un
taux de denaturation de 20 a 40%. La destruction du lysozyme est aussi plus grande en
presence de glucose lorsque Ton compare a 1’echantillon de controle. L ’etendue de la
glycosylation est egalement plus grande avec les echantillons soumis au traitement de
forte pression, dormant une glycosylation de 60% apres 8 heures d ’exposition au
traitem ent, mais diminuant a environ 40% par la suite. L ’irradiation sous champ microondes a donne environ 40% de glycosylation pour les echantillons de lysozyme apres 20
m inutes d ’irradiation.
D ’autre part, le BSA s ’est avere plus susceptible aux dommages lors de son
exposition au traitement sous forte energie. Les echantillons de BSA se sont denatures
d ’avantage que ceux du lysozyme, soit ju sq u ’a 80% lors de longues periodes
d ’expositions, mais dans tous les traitements, le glucose a semble agir comme un agent
protecteur, contrairement au cas du lysozyme. L ’etendue detectee de la glycosylation fut
egalement m inimale, variant entre 8 et 20%.
Une etude de faisabiiite pour 1’analyse des proteines giycolysees par pyrolyse en
GC/M S fut egalement examinee. Prenant avantage de la form ation d ’un m arqueur
diagnostique, le 2,3-dihydro-3,5-dihydroxy-6-m ethyl-4H-pyran-4-one, sous pyrolyse de
proteines giycolysees, 1’intensite du pic fut utilisee pour correler 1’etendue de la
glycolysation. On a trouve que 1’intensite de ce pic dans le pyrogram m e des lysozymes
giycolysees augmentait de fapon lineaire avec les temps d ’incubations plus longs et
consequemment avec le nombre de sucres presents dans les lysozymes glycolyses. De
plus, 1’utilisation des pyrogram mes comme em preintes uniques, a permis d ’evaluer
1’etendue des changem ents structuraux entre les proteines m odifiees et les proteines nonmodifiees.
IV
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ACKNOWLEDGEMENTS
Believe it or not, this is the section that I have been thinking o f w hat to put down
since I started my master studies. I really want to express my thanks and deep gratitude
to everyone who provided advice, supervision, help, encouragement and support to allow
me to get to this point. It has been a team effort in completion o f this work.
First o f all, I would like to express my deep gratitude to my supervisor, Dr. V.A.
Yaylayan. His advice and guidance were crucial to my thesis. I sincerely appreciate the
time and thoughts given by him, in which, m any o f his invaluable advice, ideas and
comm ents have been included in this thesis. His help was always there when needed.
And m ost o f all, his patience and encouragement made m y life way easier and more
pleasant throughout my graduate study.
Dr. H. Ramaswamy, as m y co-supervisor, for allowing me to have access to the
high pressure equipment. He also gave me the chance to be the teaching assistant in two
o f his courses, and provided helpful guidelines in how to m ake the class more dynamic,
which I find it very valuable in training m yself to be more organized and expressive. In
addition, thanks to the team o f graduate students in the pilot plant as well, who took their
time to assist me to operate the high pressure equipment.
Dr. J. I. Boye, who participated in meetings and gave valuable comments on the
results o f m y experiments. But I should have thanked her two years ago when I did a
summer project at St-Hyacinthe. W hat she taught me at that time m ade me very well
prepared when I started my study, and as well, when I come out to work in the future.
Special thanks go to Dr. F. K. Yeboah who provided constructive suggestions and
comments on the results o f my experiments, that were decisive.
Throughout these two years in achieving my m aster degree, the continuous
support and encouragement from my fam ily m ade my dreams come true. Distance-wise
is far, but m entally close with my m om and other family mem bers who are in Hong
V
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Kong, and to m y sister, aunty M im y and uncle, and other relatives who are in. Montreal,
thank you for supporting and sustaining m e and thanks dad, 1 miss you still.
Tim e in the lab would be very m uch different without Andre, Luke and Eva. The
fun talks and laughs created a very joyful and comfortable atm osphere in the lab. Special
thanks to Andre for teaching me how to do pyrolysis properly. Thanks to all o f them.
Thanks to Dr. I. Alii and Dr. B.K. Simpson who took their time in counseling and
directing m e whenever I needed help.
I was m uch stronger throughout these years because o f the soothing talks and
casual chats with Vivian and M andy. You have been true friends, thank you so much.
Thanks to FCAR for awarding m e the graduate scholarship, and to NSERC
strategic fond for funding this research.
Last but not least, thanks to Barbara, Lise, together with all other professors and
staffs in the food science department, who are always helpful.
VI
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TABLE OF CONTENTS
D E D IC A T IO N ..
................
II
A B S T R A C T .......................
.....I ll
R E S U M E ........................
....................I V
A C K N O W L E D G E M E N T S ..........................
.....V
TA BLE OF C O N T E N T S ...................................
LIST OF F IG U R E S .......
LIST OF T A B L E S ......
..........V I I
.............
.....X I
..................
XIV
1 INTRODUCTION.............................. .........................
......... ...................
1
2 LITERATURE REVIEW ........................................................................................... 4
2.1
IN TR O D U CTIO N .............................................
4
2.2
THE M AILLARD R EA C T IO N ..............................
4
2.2.1
CHEM ICAL PA TH W A Y S...........................
2.2.2
SIGNIFICANCE OF M AILLARD REACTION/GLYCATION IN
FO O D S.................
2.3
2.4
2.5
....4
8
FOOD PROCESSING AND NOVEL T E C H N O L O G IE S.....................................10
2.3.1
IN TR O D U CTIO N ..........................
2.3.2
HIGH HYDROSTATIC PRESSURE (HHP) AS A PR O C E SS................ 10
2.3.3
M ICROW AVE IRRADIATION AS A P R O C E S S ........................
2.3.4
PROS AND CONS OF M INIM AL PR O C E S S IN G ...................................... 13
10
12
M AILLARD REACTION AN D NOVEL TE C H N O L O G IE S.............................. 14
2.4.1
M AILLARD REACTIO N AND H H P .................................
2.4.2
M AILLARD REACTION AND M ICRO W A VE IRRA D IA TIO N ............ 15
M ETHODS OF DETECTING GLYCATED P R O T E IN S
14
16
3 MATERIALS AND M ETH O D S.............................................................................. 19
3.1
M A T E R IA L S
3.2
EQUIPM ENTS U S E D ............................
19
3.3
SAMPLE PR EPA R A TIO N ................................................................................
20
3.3.1
LYSOZYM E
.......................
.............................................
VII
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19
20
3.3.2
BSA
..........................
21
3.4
OVEN INCUBATION TREATM EN T...................................................
21
3.5
HH P TREATM EN T....
22
3.6
FOCUSED M ICROW AVE IRRADIATION TR E A T M E N T
3.6.1
3.6.2
3.7
L Y SO Z Y M E
BSA
........
23
...............
23
.................................................... .......................... ............... ........ - 23
ANALYSES OF THE TREATED SA M PLES ..................
3.7.1
LOW RY T E ST
3.7.2
FLUORESCAM INE ASSAY
4 R E S U L T S ..
24
.................
24
......................
25
............................................ .................................................................29
4.1
IN TR O D U CTIO N ...................................
29
4.2
RESULTS OF LYSOZYM E SA M PLES...................................
30
4.3
..........................
4.2.1
RESLTS OF LOWRY RESULTS
4.2.2
RESULTS OF FLUORESCAM INE A SSA Y S
30
............
36
RESULTS OF OF BSA SA M PLES.....................
42
4.3.1.
RESULTS OF LOW RY TESTS
...........................
42
4.3.2
RESULTS OF FLUORESCAM INE A SSA Y S................................................46
5 D ISC U SSIO N .................
5.1
50
EFFECT OF HHP AND M ICROW AVE IRRADIATION ON LYSOZYM E 50
5.1.1
LOW RY TEST ........................................................
50
5.1.2
FLUORESCAM INE ASSAY .........
5.1.3
COM PARISON OF THE EFFECTS OF THE TW O TREATM ENTS ON
.5 5
GLYCATION OF LY SO ZY M E......................
5.2
EFFECT OF HHP AND M ICROW AVE IRRADIATION ON B SA
61
............61
5.2.1
LOWRY T E S T
...........................................
5.2.2
FLUORESCAM INE ASSAY
5.2.3
COM PARISON OF THE EFFECTS OF THE TW O TREATM ENTS ON
.......................
GLYCATION OF B S A ............................................
5.3
68
72
OVERALL COM PARISON OF THE EFFECT OF DIFFEREN T
TREATM ENTS ON BSA A N D LY SO ZY M E...................................
5.4
62
FUTURE STUDIES ...............................................................................................
VIII
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73
74
6 A N A L Y SIS O F G L Y C A TED P R O T E IN S BY P Y R O L Y S IS -G C /M S ..,..........7 5
6.1
IN TRO D U CTIO N .........................
6.2
M ATERIALS AND M E T H O D S
6.2.1
6.2.2
75
.....................
78
.................
M A TE R IA LS
78
SAMPLE PREPARATION .....................................
78
6.2.2.1 INTERFERENCE OF FREE GLUCOSE W ITH PYROLYSIS
ASSAY
......
78
6.2.2.2 CORRELATION OF THE INTENSITY OF THE PYROLYSIS­
GENERATED M ARKER PEA K FROM COM M ERCIALLY
AVAILABLE GLYCATED HYM AN SERUM ALBUM IN (HSA) WITH
THE AM OUNT OF P R O T E IN
..........
79
6.2.2.3 CORRELATION OF THE INTENSITY OF PYROLYSIS-GENERATED
M ARKER PEAK FROM PREPARED GLYCATED LYSOZYM E WITH
THE DEGREE OF G L Y C A TIO N ............................
6.2.3
6.3
M ETH O D S.........................
80
RESULTS AND D IS C U S S IO N
6.3.1
79
.............
81
INTERFERENCE OF FREE GLUCOSE W ITH PYROLYSIS
A S S A Y ........................
6.3.2
...81
ESTABLISHING THE LIM IT OF DETECTION OF M ARKER IONS
FOR GLYCATED HUM AN SERUM A L B U M IN
6.3.3
PREDICTION OF THE EXTENT OF GLYCATION IN GLYCATED
LYSOZYM E W ITH PYROLYSIS A S S A Y .....
6.3.4
........................ 86
..................
86
INTRODUCTION TO THE CONCEPT OF SIM ILARITY INDEX TO
ASSESS STRUCTURAL CHANGES IN PROCESSED PROTEINS.... 87
6.4
C O N C L U SIO N
7 G E N E R A L C O N C L U S IO N
................................................................
.....................
A P P E N D IC E S .
A1
A2
............................................... 96
.....
TABLES OF RAW DATA OF LYSOZYM E
89
97
.........................
97
A l .l
LOW RY TEST OF LY SOZY M E SA M PL E S.................................................97
A 1.2
FLUORESCAM INE ASSAY OF LYSOZYM E S A M P L E S .....................101
TABLES OF RAW D A TA OF BSA
........................
IX
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105
A2.1
LOW RY TEST OF BSA SA M PLES.....
A2.2
FLUORESCAM INE ASSAY OF BSA SAMPLES
A3
..........
105
.......
108
SAMPLE CA LCU LATIO N............................
A3.1
Ill
A N EXAM PLE FO R CALCULATING THE AM O UN T OF BSA
PRESENT IN THE SAMPLES THAT G ELLED
................. 111
A3.2
STEPS IN CALCULATING THE EXTENT OF G L Y C A TIO N .............. I l l
A3.3
EQUATIONS FO R THE CA LCU LA TIO N S...
A 3.4
AN EXAM PLE IN CALCULATING THE EXTENT OF
.......................
G LYCA TION......................
R E F E R E N C E S ..
112
113
.......................................................................
X
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114
L IS T
OF FIGURES
Figure 2.1
Early stage o f M aillard reaction between a carbonyl group and an amino
group and formation o f Amadori compound, showing partial
structures
Figure 2.2
..............
...6
Reaction pathways o f the advanced and final stages o f M aillard reaction
adapted from Hodge (1 9 5 3 )..........
Figure 5.1
...7
Percentage o f water soluble lysozyme rem ained in the samples upon being
subjected to regular incubation at 30°C over a period o f 12 days, as
compared to the initial amount o f protein at day 0 ...............
Figure 5.2
51
Percentage o f water soluble lysozyme rem aining in the samples (that
contained 0.01% sodium azide) upon being subjected to regular
incubation at 30°C over a period o f 12 days, as com pared to the initial
amount o f protein at day 0
Figure 5.3
.....................
52
Percentage o f soluble lysozyme rem ained in samples upon being
subjected to HHP from 8 hours to 48 hours at 30°C, as compared to the
initial amount o f protein at day 0 ..........................
Figure 5.4
54
Percentage of soluble lysozyme rem aining in samples subjected to
m icrowave irradiation from 10 minutes to 60 m inutes at 50°C, as
com pared to the initial amount o f protein at day 0 .........................
Figure 5.5
54
Correlation o f the extent o f glycation o f lysozym e-glucose m ixture that
have been subjected to regular incubation at 30°C over a period o f 12 days
when (a) 0.01% sodium azide was absent; and (b) 0.01% sodium azide
was present. Both are compared to the initial extent o f glycation o f the
day 0 sam ple...................
Figure 5.6
57
Extent o f glycation o f lysozym e-glucose m ixture that have been subjected
to (a) 400 M Pa o f HHP at 30°C over a period o f 48 hours; and (b) regular
incubation at 30°C over a period o f 12 days, both are com pared to the
initial extent o f glycation o f the day 0 sam ple.................................
Figure 5.7
58
Extent o f glycation o f lysozym e-glucose m ixture (a) subjected to focused
m icrowave irradiation at 50 °C over a period o f 60 m inutes; and (b)
XI
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subjected to regular incubation at 30°C over a duration o f 12 days, both
are compared to the initial extent o f glycation o f the day 0 sam ple..... ...60
Figure 5.8
Correlation o f the extent o f glycation o f lysozyme-glucose mixture versus
the duration o f microwave irradiation at 5 0 ° C .........
Figure 5.9
.60
Percentage o f water soluble BSA rem ained in the samples upon being
subjected to regular incubation at 30°C over a period o f 12 days, as
compared to the initial amount o f BSA at day 0 .
Figure 5.10
.......
Percentage o f soluble protein o f BSA samples subjected to HHP at 30°C
as compared to the initial amount o f the protein at day 0 .............
Figure 5.11
...64
....66
Percentage o f soluble BSA in samples subjected to focused microwave
irradiation from 10 minutes to 60 minutes at 50°C, as compared to the
initial amount o f the protein at day 0 ........................
Figure 5.12
68
Correlation o f the extent o f glycation o f BSA-glucose m ixture versus time
o f incubation at 30°C relative day 0 B SA-glucose sam ple........................ 69
Figure 5.13
Correlation o f the extent o f glycation o f BSA-glucose mixture versus the
time o f (a) HHP treatm ent at 30°C over a period o f 48 hours; and (b)
regular incubation at 30°C over a duration o f 12 days, both are compared
to the initial extent o f glycation o f the day 0 BSA-glucose sample
Figure 5.14
69
Extent o f glycation o f BSA-glucose m ixtures (a) subjected to microwave
irradiation at 50°C over a period o f 60 minutes; and (b) subjected to
regular incubation at 30°C over a period o f 12 days, both are compared to
the initial extent o f glycation o f the day 0 BSA-glucose sam ple............... 71
Figure 5.15
Correlation o f the extent o f glycation o f BSA-glucose mixture versus the
tim e under m icrowave irradiation...........................................................
Figure 6.1
72
The degradation o f Amadori rearrangem ent product upon pyrolysis with
the production o f the ion at m /z 144..................................
Figure 6.2
..77
Pyrograms o f glucose showing (a) total ion current and (b) Extracted Ion
chrom atograms (m/z 144 and m /z 126). Peaks containing the M + ion at
m /z 144 and the ion from HM F at m /z 126 are designated by I and H
respectively. .......
...82
XII
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Figure 6.3
Pyrograms showing the total ion count o f (a) HSA; (b) GHSA; (c) HSA
with glucose; (d) lysozyme; (e) lysozyme with glucose. Peaks containing
the M + ion at m/z 144 are designated by I in the pyrogram s..................8 3
Figure 6.4
Extracted Ion chromatograms (m/z 144 and m/z 126) o f (a) HSA; (b)
GHSA; (c) HSA with glucose; (d) lysoyzme; (e) lysozyme with glucose.
Peaks containing the M + ion at m /z 144 and the ion from HMF at m/z 126
are designated by I and H respectively...............
84
Figure 6.5
Overlaid chromatograms o f HSA and G H SA ................................. ...........9 0
Figure 6.6
Pyrograms showing the total ion current o f the lysozyme-glucose
incubation samples for (a) 2 days; (b) 5 days; (c) 10 days; (d) 14 days.
Peaks containing the M + ion at m/z 144 are designated by I
Figure 6.7
...........91
Extracted Ion chromatograms (m/z 144) o f the lysozyme-glucose samples
being incubated for (a) 2 days; (b) 5 days; (c) 10 days; (d) 14 days. Peaks
containing the M + ion at m/z 144 is designated by I ............................. .....92
Figure 6.8
Correlation o f the time o f the lysozyme samples being incubated at 50°C
over a period o f 14 days versus the signal intensity o f the diagnostic peak
after deconvolution.............................................................................................. 93
Figure 6.9
Correlation o f the average num ber o f sugar m olecule attached per
m olecule o f lysozyme versus the signal intensity o f the diagnostic peak
after deconvolution........................................................
Figure 6.10
...93
Pyrograms showing the total ion current of the lysozyme samples that had
been subjected to (a) 8 hours o f HHP and (b) 10 m inutes of focused
m icrowave irradiation. Peaks containing the M + ion at m/z 144 and the ion
from HMF at m/z 126 are designated by 1 and H respectively
XIII
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.....94
LIST OF TABLES
Table 3.1
Dilutions done o f the treated samples for analysis te sts.
Table 3.2
The amount o f gel recovered and BSA content o f HHP treated
sam ples.
Table 3.3
26
.............
..27
Amount o f water, SDS and the calculated BSA content in the microwave
treated sam ples.
Table 4.1
.......
...................................................
...28
Normalized absorbance and calculated protein content o f incubated
lysozyme samples that did not contain sodium azide..................
Table 4.2
30
Norm alized absorbance and the calculated protein content o f the lysozyme
samples that contain glucose and 0.01% sodium azide being subjected to
regular incubation from day 0 to day 1 2 ...........
Table 4.3
31
Norm alized absorbance and the calculated protein content o f the control
samples o f lysozyme w ith 0.01% sodium azide being subjected to regular
incubation from day 0 to day 12...............
Table 4.4
32
Norm alized absorbance and the calculated protein content o f the lysozym e
samples being subjected to HHP treatm ent from 8 hours to 48 hours at
400 M Pa
Table 4.5
................
33
Norm alized absorbance and the calculated protein content o f the lysozyme
samples that contained glucose being subjected to m icrowave irradiation
from 10 minutes to 60 m inutes
Table 4.6
............
34
Norm alized absorbance and the calculated protein content o f the control
samples o f lysozyme being subjected to m icrowave irradiation from 10
minutes to 60 m inutes
Table 4.7
......................
35
The calculated extent o f glycation or denaturation o f the lysozyme
incubated samples without sodium azide from day 0 to day 12............. ....36
Table 4.8
The calculated extent o f glycation o f lysozyme samples that contained
glucose and 0.01% sodium azide being subjected to regular incubation
from day 0 to day 12......
37
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Table 4.9
The calculated extent o f denaturation o f control samples o f lysozyme that
contained 0.01% sodium azide when being subjected to regular incubation
from day 0 to day 1 2 ...................
Table 4.10
....38
The calculated extent o f glycation or denaturation o f lysozyme samples
being subjected to HHP from 8 hour to 48 hours at 400 M P a
Table 4.11
.......39
The calculated extent o f glycation o f lysozyme samples that contained
glucose being subjected to microwave irradiation from 10 minutes to 60
m inutes
Table 4.12
.............
..40
The calculated extent o f denaturation o f lysozyme controls being
subjected to m icrowave irradiation from 10 m inutes to 60 m inutes
Table 4.13
Norm alized absorbance and calculated protein content o f BSA samples
subjected to regular incubation
Table 4.14
41
...............................
42
Norm alized absorbance and the calculated protein content o f BSA
samples that were being subjected to HHP from 8 hours to 48 hours at 400
M P a.........................
Table 4.15
43
Norm alized absorbance and the calculated protein content o f BSA
samples containing glucose that were being subjected to m icrowave
irradiation from 10 m inutes to 60 m inutes
Table 4.16
.....................
44
N orm alized absorbance and the calculated protein content o f BSA control
samples that were being subjected to m icrowave irradiation from 10
minutes to 60 m inutes...................................
Table 4.17
45
The calculated extent o f glycation or denaturation o f BSA samples being
subjected to incubation from 0 to 12 days.......................................................46
Table 4.18
The calculated extent o f glycation or denaturation o f BSA samples that
were being subjected to HHP from 8 hours to 48 hours at 400 M Pa
Table 4.19
47
The calculated extent o f glycation o f BSA samples which contained
glucose when being subjected to microwave irradiation from 10 minutes
to 60 m inutes
Table 4.20
......................
48
The calculated extent o f denaturation o f BSA controls w hen subjected to
m icrowave irradiation from 10 minutes to 60 m inutes.............................. .49
XV
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Table 6.1
The constituent o f the samples duly prepared for pyrolysis. Lysozyme
sample treated with 8 hours o f HHP is designated by HHP, and sample
treated with 10 m inutes; o f m icrowave irradiation is designated by
M W ............................
Table 6.2
...............8 0
The weight o f GHSA and silica gel in the m ixture prepared for
pyrolysis..............
Table 6.3
...80
Amount o f GHSA pyrolyzed and the corresponding signal intensity o f the
diagnostic peak after deconvolution
Table 6.4
..........
95
Com parison o f treated lysozyme samples with lysozym e/glucose based on
%purity and %fit, and the corresponding Similarity Index ( S I ) ................ 95
Table A l . l
Absorbance at 540nm o f the lysozyme incubation samples with no sodium
azide
Table A1.2
.......
97
Absorbance at 540 ran o f the lysozym e incubation samples with 0.01%
sodium azide..................
Table A1.3
98
Absorbance at 540 rnn o f the lysozyme samples that have been subjected
to HHP at 400 M P a ...
Table A1.4
................................................... ......99
Absorbance at 540 nm o f the lysozyme samples that have been subjected
to m icrowave irradiation from 10 minutes to 60 m inutes..........................100
Table A1.5
Relative fluorescence o f the lysozym e incubation samples with no sodium
azide added..................................................
Table A1.6
101
Relative fluorescence o f the lysozym e incubation samples with 0.01%
sodium azide.................................
Table A 1.7
Relative fluorescence o f the lysozyme samples being subjected to
HHP
Table A1.8
.......................................................
103
Relative fluorescence o f the lysozyme samples that have been subjected to
microwave irradiation
Table A2.1
102
......
104
Absorbance o f the BSA incubation samples from 0 to 12 days at 540
nm
.............
105
Table A2.2
Absorbance at 540 nm o f the BSA samples being subjected to HHP. ...106
Table A2.3
Absorbance at 540 nm o f the BSA samples that have been subjected to
m icrowave irradiation.
..................................................107
XVI
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Table A 2.4
Relative fluorescence o f the BSA incubation sam ples.
Table A 2.5
Relative fluorescence o f the BSA samples being subjected to H H P
Table A 2.6
Relative fluorescence o f the BSA samples that have been subjected to
microwave irradiation.
........
.........
XVII
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108
109
..110
1 INTRODUCTION
T he M aillard reaction, which involves the interaction o f amino acids or proteins
w ith reducing sugars, has been the subject o f investigation by food scientists and by the
food industry, due to its effect on over-all quality attributes o f processed food. This
reaction w hich is well known to contribute to deterioration o f food quality during
processing and storage (Reynold, 1965; Dworschak, 1980), at the same time, is also
known to im part desirable flavor and color, as well as texture to some processed foods.
This dual role o f M aillard reaction in food processing makes it necessary to study the
m eans o f its control.
Thermal treatment has always been used as a means o f processing food to
generate desirable flavors and aromas. However, heat processing generally causes
degradation o f thermally sensitive nutrients, vitamins, colors, flavors and texture
(Khamrui and Rajorhia, 2000). Novel technologies with m inimal processing o f food,
such as high hydrostatic pressure and m icrowave irradiation, can overcom e some o f these
undesirable side effects o f high tem perature processing.
Application o f high hydrostatic pressure to food systems has been used for
decades as a means to preserve food, and it is only in the last 15 years that scientists
started to investigate the effects o f high pressure on chemical reactions, specifically
M aillard type reactions. With an increase in the activation volume due to the formation
o f volatiles and other small m olecules that are important for flavor production, the rate o f
the final stage o f reaction is retarded or suppressed with the application o f high pressure
1
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(Tam aoka et at.,
1991; Issacs and Coulson,
1996; Bristow and Issacs,
1999;
Schwarzenbolz et a l, 2000); while the rate o f the formation o f the initial condensation
products (Amadori products) is accelerated. This important property can be exploited to
generate glycated proteins to m odify the functional properties o f different food related
proteins.
M icrowave ovens on the other hand, have always been used as a means o f
reheating foods by the consum er (Schiffmann, 1994). Due to its property o f high degree
o f penetration and rapid heat transfer, food manufacturers have been trying to exploit
ways to process foods such as tempering o f frozen fish or m eat and drying o f fruits and
vegetables (Ramaswam y and Van de Voort, 1990; International Review, 1997).
M icrowave energy can also be used to carry out chemical reactions such as M aillard
reaction (Yaylayan, 1996). M aillard reaction between tofu and glucose was observed
with a considerable weigh loss and decrease in solubility o f tofu upon m icrowaving
(Kaye et al., 2001).
In foods, proteins play important functional role in dictating the overall
perception o f the food products. Some o f the functional properties include solubility,
viscosity, water binding capacity, gelation ability, elasticity, etc to name but a few.
Glycated proteins, with controlled attachm ent o f glucose to the proteins, can lead to a
decrease or an increase in solubility (Kato et al., 1978; H anda and Kuroda, 1999) or
enhancement o f emulsifying properties, with a decline in gel strength upon the
2
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occurrence o f M aillard reaction (Miyaguchi et al., 1999). Therefore, depending on the
type o f product to be manufactured, the extent o f glycation should be controlled.
This thesis will investigate the effect o f high hydrostatic pressure and microwave
irradiation on glycation o f two common food proteins, lysozyme and BSA, using Lowry
test (Lowry, 1951) and fluorescamine assay (Yaylayan et a l, 1992) to determine the
amount o f soluble proteins and the extent o f glycation. In addition, a fast and convenient
m ethod based on Pyrolysis-GC/M S will be developed to analyze glycated proteins.
3
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2 L IT E R A T U R E R E V IE W
INTRODUCTION
2.1
M aillard reaction, or non-enzymatic browning, is a chemical interaction that occurs
widely in food and biological systems. It refers to the reactions initiated by the
interaction betw een an amino group and an a-hydroxycarbonyl m oiety o f a reducing
sugar (Yaylayan, 1997). In food systems, the prim ary source o f free amino compounds
comes from the amino acids in the proteins; while reducing sugars are the primary source
that supplies the carbonyl groups. This reaction governs the formation o f color and flavor
in foods, as well as the properties o f the proteins involved. However, the M aillard
reaction, also known as glycation when proteins are involved, is not ju st a single reaction,
but a cascade o f com plex reactions depending on the corresponding precursors and the
reaction conditions (W eenen et a l, 1997). Since the discovery o f browning between
amino-carbonyl compounds by M aillard in 1911, the chemistry o f the M aillard reaction
has been extensively studied, as well as the different aspects in which M aillard reaction
plays a role, specifically in food and physiological systems. A b rief summary o f
glycation and the chemistry of M aillard reaction will be presented in this review.
2.2
THE MAILLARD R E A C T IO N
2.2.1
CHEMICAL PA TH W A Y S
The complexity o f the M aillard reaction lies in the fact that the reaction is
difficult to stop once it is initiated. It is accom panied with the formation o f a multitude o f
compounds in minute amounts taking place by side reactions and obscure pathways.
M aillard reaction can occur even at m oderate room tem peratures, and the rate o f reaction
4
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is accelerated with increasing temperatures. The chemical pathways can broadly be
divided into the early, intermediate and the final stages (Hodge, 1953). In the early stage
o f the M aillard reaction, the carbonyl group undergoes condensation with the amino
group w ith the loss o f a water molecule, forming the Schiff base, glycosylamine (Figure
1.1). This compound being unstable, will then undergo subsequent isomerizatioxis to a
m ore stable form. Amadori compound is form ed when the amine rearranges w ith aldose
to an amino ketose, while Heyns product is formed w hen the amine rearranges with
ketose to form an amino aldose (Ledl, 1990). Up to this point, reactions are reversible in
aqueous solutions, since the glycosylamine can be hydrolyzed into its parent compounds
(Namiki, 1988). However, the pH o f the system changes as the reactions proceed with
the form ation o f different products. Depending on the pH o f the system, different
reaction pathways will take precedence in the advanced and final stages o f the M aillard
reaction, leading to the form ation o f nitrogenous polymers and co-polymers, known as
m elanoidins (Figure 1.2). With an alkaline environm ent having a pH greater than 7, chain
fragm entation o f the Schiff base and the Am adori compound will occur, leading to the
form ation o f 2- and 3-carbon fragments which will undergo further irreversible reactions
to form the brown pigments. W ith a more acidic or a neutral environm ent having a pH
less than 7, deoxyosones will be formed by eliminating the original amine from the
Am adori compound. The deoxyosones are very reactive intermediates and will undergo
cyclization or dehydration to form higher m olecular weight compounds, generating
flavor and color (Rizzi, 1994).
5
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H C =0
HNR-CHOH
I
j
RNH2
CHOH
I
CHOH
HC=N-R
- H20
^
CHOH
I
■*—
CHOH
1
CHOH
aldose
I
addition com pound
(aldehyde form)
CHOH
Schiff base
aldoosylam ine
ch2 nhr
CH NHR
c=o
C-OH
CHOH
CHOH
k e to form
enol form
1 -amino-1 -deoxy-2-ketos®
Amadori C om pound
Figure 2.1
Early stage o f M aillard reaction betw een a carbonyl group and an amino
group and formation o f Amadori compound, showing partial structures.
6
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Amadori reanflgenisi product (ARP)
l^mii^Meoxyibtose
! +2H
+a
IM F or furfural
AidiminesandKetimes
Melanoidins (brown nitrogenous polymers)
Figure 2.2
Reaction pathways o f the advanced and final stages o f M aillard reaction
adapted from Hodge (1953).
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2.2.2
S IG N IF IC A N C E O F M A IL L A R D R E A C T IO N /G L Y C A T IO N IN FO O D S
Proteins dictate the corresponding nutritional quality and the functional properties
in foods w ith the type and amount o f amino acids present and the corresponding
sequence o f the am ino acids which form the structure o f the proteins. M aillard reaction is
a reaction that can occur m ainly between the y-lysyl groups o f the proteins and the
aldehyde groups o f reducing sugars, and it occurs even under the m ildest conditions.
Therefore, with the onset of the reaction, availability o f lysine will be significantly
reduced (Hurrell, 1990). Proteins that have reducing sugars attached to them are termed
m odified proteins, and they exhibit different functional properties as compared to the
original unm odified proteins. As the reaction proceeds to the advanced and final stages,
free lysine could be destroyed, as well as other essential amino acids such as histidine,
arginine, tryptophan, cysteine etc, in which they will undergo further reactions with
prem elanoidins. Thiamine, a vitamin, has an amino group which can also take part in
M aillard reaction if present in the food, and can possibly have its bioavailability reduced.
Despite the effects on the nutritional quality o f the foods, M aillard reaction is an
important chemical reaction to mankind, because it is responsible for generating the
flavor and color. With the modifications o f proteins, their functional properties can also
be changed. Different proteins with different glycation conditions produce different
functional properties. Soy protein isolates, for example, when glycated for 3 days showed
improved solubility, emulsifying stability and foam expansion, while showing slight
decrease in the fat binding and water holding capacity (Boye et a t, 2002). Solubility o f
the bovine serum album in (BSA)-glucose mixture and the ovalbum in-giucose complex in
8
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their dry state decreased as the incubation period lengthened (W atanabe and Sato, 1980,
Yeboah e t a l, 1999); emulsifying properties, gelling and foaming properties o f the
ovalbum in-dextran complex were improved as M aillard reaction proceeded (Nakai,
2000). Functional properties o f the proteins in food therefore play a significant role in the
perception o f the food products. Controlled glycation can therefore generate glycated
proteins w hich can be suitable for producing food products with different sensory and
functional properties.
Flavor, which refers to a combination o f smell and taste, is the other area that
governs the overall perception o f food products, and is the m ost important factor o f all.
Since the discovery o f fire by mankind, hum an beings used therm al energy to cook food
to acquire the desired flavor, texture and color. Application o f heat to bread-baking,
browning o f meats and others at the suitable tem peratures and for the right duration o f
time can generate desirable flavors; while on the other hand, over-processing the product
can generate unacceptable flavors. The aroma compounds are the smell chemicals that
are generated from the M aillard reaction, and are low m olecular weight compounds that
are usually volatile. They could be formed by first producing lower m olecular weight
compounds such as pyruvaldehyde from retro aldol reactions o f deoxyosones obtained
from the initial stage o f M aillard reaction. They will then react with the amino acids in
the systems to form flavor voiatiles (Rizzi, 1994). Other pathw ays in generating arom a
compounds include cyclization or dehydration o f deoxyosones. Compounds such as
furanones could be generated, which have a distinct smell by itself, but they could also
undergo further reactions to generate different flavor, such as reacting with the sulfur in
9
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the system (if present) and generate a wide range o f aromas. Because o f the numerous
possible pathw ays that could generate different aromas and taste compounds, M aillard
reaction is very complex and complicated. Since this review is on the glycation o f food
proteins, this section will m ainly deal with the formation o f Amadori compounds when
the proteins and sugars are processed using novel technologies.
2.3 F O O D P R O C E S S IN G AND N O V E L T E C H N O L O G IE S
2.3.1
IN T R O D U C T IO N
Cooking is a form o f thermal processing that is used to add value to the raw
m aterials in order to produce desirable flavors, aromas and texture, and to transform the
raw m aterials to an edible and safe-to-consum e form. The food industry utilizes similar
principle to commercially produce food products for consumption in a ready-to-eat form
like m ilk or foods with a longer shelf life like canned foods. There has been increasing
interest by the food industry to investigate alternate lower-cost technologies to process
foods with minimal damage to the food components.
2.3.2
H IG H H Y D R O ST A T IC P R E S S U R E (H H P ) AS A PR O C E S S
The use o f high hydrostatic pressure (HHP) on foods has been around for over a
century. Food industry utilizes the ability o f high pressure to inactivate micro-organisms
to process food to be safe for consum ption and have extended shelf life. A typical high
pressure system consists o f a high pressure cham ber with closure in which samples or
packages are loaded; a pressure generating system to generate pressure either by direct or
indirect compression; and a tem perature control device to m onitor the tem perature o f the
10
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pressure
chamber either in the form o f sensors for large-scale production or
therm ocouples for laboratory-scale equipment (Ting et a l, 2002). Physical-chemical
properties o f water such as phase transition and density can he m odified upon the
application o f pressure, and can therefore induce protein denaturation and modification
o f biopolym ers in food affecting properties such as gelling, susceptibility to enzymatic
degradation, etc. (Knorr, 1994). In addition, losses in nutrients and quality o f the
products w ith ultra high pressure can be minim ized as com pared to thermal processing
because the process could be carried out at ambient temperatures, depending on the types
o f m icro-organism s to be destroyed or inactivated. Vegetative cells and yeasts are
pressure-sensitive, and they can be destroyed at ambient tem peratures with above 100
M Pa o f pressure (Hoover et al., 1989); bacterial spores, on the other hand, are more
resistant to pressure and requires about 600 M Pa o f pressure at elevated temperatures
betw een 45-60°C (Hayashi, 1992). It m ust be noted that with such high pressure, food
products will not be deformed and will retain their shape after being processed, since
pressure is being applied uniformly from all directions on the products. In other words,
the operation o f high pressure is independent o f size and geometry o f the product to be
pressurized.
Although the capital cost for installing the high pressure equipm ent is usually
high (M eyer et a l, 2000), nonetheless, because o f its short process times and high quality
o f foods generated, the use o f HHP as a process which has been known for around 100
years, starts to attract the attention o f food m anufacturers to process food in the last
decade, especially those in Japan which demand high quality foods.
11
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2.3.3
MICROWAVE IR R A D IA T IO N AS A PROCESS
M icrow ave energy is the electromagnetic radiation that lies between infrared
irradiation and radio waves and has a frequency range between 300 M Hz to 300 GHz
(Jaiichem, 1998). It was discovered Dr. P. Spencer in 1946 while testing a vacuum tube
called m agnetron (Gailawa, 2002). M icrowave energy can be converted into heat by
either dipole rotation or ionic conduction. In other words, the presence o f either dipolar
or ionic m olecules can interact with the electric field and generate heat (Yaylayan, 1996).
Because m icrowave irradiation has relatively long wavelengths, it has a high degree o f
penetration and can therefore rapidly transfer heat throughout the m olecules to be
irradiated. Hence, m icrowave processing can save more time in processing as compared
to therm al processing.
Invention o f the domestic m icrowave ovens did not gain popularity for use at
homes until the last two decades when more wom en entering the work force, had
increased dem and for a more convenient tool to reheat food rapidly (Ramaswamy and
Van de Voort, 1990). The food industrial sector also took this opportunity to investigate
the potential o f exploiting microwave irradiation as being an economical means o f
processing foods because o f its rapid heating characteristics. Drying, thaw ing and
thermal processing are some o f the common m icrowave applications in the food industry
today (International Review, 1997; Ram aswamy and Van de Voort, 1990). M icrowavedried tarhana (a wheat flour-yogurt mixture) samples exhibited higher overall sensory
rating and color acceptability as com pared to freeze-dried and tunnel-dried samples
(Hayta et a l, 2002). Thawing o f m odel frozen foods using m icrow ave also showed a
12
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seven-fold reduction in time as compared to convective thawing at ambient temperature
(V irtanen et a!., 1997). Industrially, m icrowave tempering for converting hard-frozen
foodstuffs into a workable, usable and tem pered form which are not necessarily thawed
com pletely is necessary for frozen food industry for further food processing, in which,
the tem pered foodstuffs did not show any quality7 loss (Decareau, 1985). Focused
m icrow ave heaters are also used to pasteurize and process foods such as dairy products
and nutritional products like protein beverages. A focused m icrowave system differs
from the dom estic m icrowave oven in that the m icrowave irradiation is being delivered
to the object to be irradiated directly in a beam, instead o f allowing the Irradiation beams
to bounce back and forth on the walls o f the m icrowave oven before hitting the sample.
M icrowaves in this case are being focused throughout the volume o f the product,
allowing for more uniform and instantaneous heating, with m inimal loss in flavor and
color (Clark, 2002).
2.3.4
PROS AND CONS OF M IN IM A L PROCESSING
M inimal processing offers the advantage o f keeping the food safe and extending its
shelf life. Product quality can as well be improved in retaining more o f the thermallysensitive nutrients, colors, and flavors as com pared to regular thermal processing.
However, the lack o f M aillard reaction in producing the desired colors, flavors and
textures to the food product as com pared to thermal processing poses a m ajor
disadvantage to the consumer acceptance.
13
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2.4
MAILLARD R E A C T IO N AND N O V E L T E C H N O L O G IE S
2.4.1
MAILLARD REACTION AND H H P
O ther than being used for destroying micro-organisms, HHP can influence
chem ical reactions depending on the changes in the activation volumes that occur. A
kinetic study by Tam aoka et a l (1991) concerning the effect o f chemical reaction under
HHP show ed that when the activation volume decreases with the production o f smaller
num ber o f m olecules having less activation volume (3.9-8.9 mL/mmol), high pressure
favors the M aillard reaction. On the other hand, when the reaction will generate more
molecules, no m atter whether they are small in size or not, they will have a greater
activation volume (12.8-27.0 m L/m mol) and will not be favored. Therefore, in the early
stage o f M aillard reaction, formation o f the Am adori compound from the amino acids or
proteins w ith the carbonyl groups o f reducing sugars (resulting in only one ketosam ine
compound) will be favored by high pressure, w hile the later stage o f browning that forms
num erous volatile compounds and color will not. Okazaki et al. (2001) used white sauce
m ade from milk, flour and butter as a model food to study the effect o f HHP on
browning. It showed that the white sauce that was heated without HHP treatm ent
browned more significantly than the white sauce being subjected to pressurization with
heating to 115°C for 30 minutes. This further confirms that HHP suppresses the Amadori
product from proceeding to the browning reaction which happens in the advanced and
final stages o f the M aillard reaction. Issacs and Coulson (1996) also suggested the
accumulation o f Am adori product under HHP due to the retardation in forming advanced
M aillard reaction products that come from the degradation o f the Am adori rearrangem ent
product (ARP).
14
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2.4.2
MAILLARD R E A C T IO N AND M IC R O W A V E IR R A D IA T IO N
Although microwave irradiation can serve a wide variety o f uses in the food
industry, it is rarely used to cook food because o f its incapability to generate M aillard
flavors. The presence o f the ionic or dipolar molecules in food in which microwave
irradiation has an effect are usually in the form o f water within the food matrix. In other
words, w hen the food is being microwaved, the highest tem perature is not at the surface,
resulting in the incapability to form a crust, leading to reduced flavor and less color
form ation (Lingnert, 1990). This also explains why the general public m ainly use
m icrowave oven to reheat leftovers o f food or defrost frozen m eat, and rarely use it for
cooking to generate the desired flavors for consumption (1997 Consum er Survey
Summary).
To overcome the lack o f flavor and color developm ent o f m icrowave food
products, different strategies have been attempted. Some aim ed at the modification o f the
cooking environment by using absorbing susceptor sheets or special microwave
browning pans (Van Eijk, 1994); some combined microwave cooking with thermal heat
(Eke, 1997); some added commercial flavorings or special coatings to food product prior
to m icrowaving to produce the desired aromas and color (Reineccius and W horton, 1990).
Food scientists also tried to investigate the possibility o f generating desired flavor
compounds with the microwave technology. A reaction system o f proline and glucose
was used to compare the effects o f thermal and m icrowave m ediated reactions on
M aillard reaction (Parliament, 1993). The GC/M S analysis o f the samples revealed that
the volatiles produced from the therm ally treated systems were generally the same
qualitatively as that from the m icrowave treated systems, but the m icrow ave samples had
15
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larger quantities o f carbohydrate decomposition products while the thermally treated
samples had larger quantities o f N-heterocyclic compounds. Yaylayan et a l (1994) also
initiated
M aillard
reaction
under
microwave
conditions
by
having
sufficient
concentrations o f the reactive M aillard precursors in the m odel systems consisting o f
different am ino acids with glucose. However, no studies have been reported on protein
glycation by m icrowave irradiation.
2.5
M E T H O D S OF D E T E C T IN G G L Y C A T E D PROTEINS
The detection o f glycated proteins formed from M aillard reaction is made
possible by a num ber o f chemical and analytical methods. Chem ical methods assay the
sugar-bound protein or protein-bound sugar via chemical reactions, w ith the detection o f
the product form ed either colorimetrically or fluorometricaily. In the thiobarbituric acid
(TBA) m ethod, the glycated protein is heated in oxalic acid to release the sugar that was
previously attached. The sugar-free protein is then rem oved by acid precipitation, while
the sugar that rem ained is converted into 5-hydroxym ethylfiirfural (HMF). The
condensation product o f HMF with TBA can be detected by spectroscopy or analytical
methods such as high pressure liquid chrom atography (HPLC) (Fluckiger and Gallop,
1984; Furth, 1988). Borohydride reduction works by reducing the C = 0 and C=N o f ARP
with sodium borohydride under alkaline conditions (Mendel, 1996; David et al,, 1998),
followed by subsequent methods in quantifying the amount o f glycated proteins that are
present. Siciliano et al. (2000) digested the reduced protein with trypsin and analyzed the
resulting m ixture with m atrix assisted laser desorption ionization (M ALDI) mass
spectrometry. The fructosamine method, or nitroblue tetrazolium (NBT) m ethod, is a
16
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com m on m ethod that is used to estimate the amount o f glycated serum protein in diabetic
patients, in which, the NBT dye reduces the glycated protein to form a bine product that
can be detected at 540 run (Forth, 1988). Other color tests for the presence o f ARP such
as ferricyanide test in which glucose reduces ferricyanide to ferrocyanide that is blue in
color (Borsook et a l, 1955). All these tests are simple, but they are very susceptible to
interference by other compounds in the system such as the presence o f free sugars.
Rem oval o f the free sugars by dialysis prior to testing could be performed, but this will
increase the overall time for the analysis.
Analytical assays based on separation o f the glycated species from the unglycated
species in the m ixture could be done by using different types o f chromatography, in
which, the protein itself is being assayed, and hence elim inating the need to separate the
protein from the unreacted sugars prior to analysis. However, m any o f these separationbased assays are highly selective. For example, phenylboronate affinity chromatography
can separate glycated proteins efficiently, provided that the sugars that are being attached
to the ARP are not phosphorylated since they will be weakly adsorbed to boronate resins,
leading to underestim ation o f glycation.
The ARP can alternatively be transform ed to compounds that fluoresce and can
be measured as such. The furosine method m easures the amount o f furosine that was
formed upon acid hydrolysis o f ARP. The furosine formed is a specific product o f lysine
ARP, and accom panying its formation is pyridosine which is also formed in minor
amounts (Furth, 1988; M arconi et al., 2002), both are separated and quantified by HPLC
at 280 nm. A non-separation fluorescence quenching assay m easures the fluorescence
17
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
generated when a fluorescein-boronic acid derivative is quenched with the glycated
protein at an excitation wavelength o f 450 ran and an emission wavelength o f 525 nm
(Blincko et al., 2000). Fluorescamine assay is another m ethod that can be used to detect
the glycated proteins. Glycated proteins are brought to alkaline conditions w ith borate
buffer (pH 8.5), fluorescamine is added to the solution and the fluorescence is measured
at an excitation wavelength o f 390 nm and an emission wavelength o f 475 nm (Yaylayan
et a l, 1992). Fluorescamine reacts with e-amino groups o f lysine to generate
fluorescence, while reducing sugars will not react. This assay measures the relative
extent o f glycation with respect to the blank that contains no protein. In addition,
prepared glycated proteins can be assayed w ithout the rem oval o f sugars, because the
assays are targeted on the protein or the ARP, and not the sugars.
In this study, the effect o f HHP and focused microwave irradiation on glycation
o f lysozym e and BSA will be investigated. The extent o f glycation and protein
denaturation that occurred upon subjecting the m odels to the high energy exposures will
be determ ined by fluorescamine assay and the Lowry test respectively.
18
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
3 MATERIALS AND METHODS
3.1
MATERIALS
Lysozym e, bovine serum albumin (BSA), D-ghicose, fluorescamine and sodium
tartrate dehydrate were all obtained from Sigma Chemicals (St-Louis, MO., USA). The
rem aining reagents used for the Lowry test were from Aldrich Chemical Company, Inc
(New Jersey, USA), Sodium dodecyl sulphate (SDS) used for solubilizing samples that
gelled
upon
processing
was
obtained
from
Aldrich
Chemical
Company,
Inc.
(M ilkwaukee, USA) and potassium tetraborate tetrahydrate used for fluorescamine assay
was obtained from Acros Organics (New Jersey, USA). W ater used throughout the study
was ultra-pure water obtained from the M illi-Q reagent grade water system (Millipore
Corp., Bedford, MA).
3.2
E Q U IP M E N T S USED
Regular incubation experiments were carried out with an Isotemp® Vacuum Oven
280A (Fisher Scientific, ON, Canada). HHP experiments were carried out in an ABB
Isostatic Press M odel # CIP42260 (ABB Autoclave System, Autoclave Engineers, Erie,
PA) with a 10 cm diam eter and 55 cm height stainless steel pressure chamber. The
equipment was rated for operation up to 414 MPa. W ater containing a 2% water soluble
oil (Autoclave Engineers, Part No. 5019, Autoclave Engineers, Erie, PA) was used as the
hydrostatic fluid in which the test packages were submerged during the pressure
treatment. Focused microwave irradiation experiments were carried out at atmospheric
pressure with a Synthewave S402 Prolabo m icrowave reactor (Fontenay-Sous-Bois,
France) with a mono-m ode M W cavity that operated at 2450 M Hz w ith power range o f
19
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0-300 W in a 12 cm3 tubular quartz reactor with irradiation being monitored by PC. The
tem perature o f reaction media was measured continuously with an IR-pyrometer, which
is an integral part o f the Synthewave 402. Lowry tests were carried out with a Beckman
UV -V isible scanning spectrophotom eter (Berkeley, CA, USA), and fluorescamine assays
were carried out with a Kontron spectrofluorometer (Kontron Instruments SFM 25
spectrofluorom eter, Zurich, Switzerland).
3.3
SAMPLE PREPARATION
Unless otherwise stated, the same sample preparation procedure as for the stock
solution was followed for all the treatments. Two control samples were used, one did not
contain any glucose and was subjected to treatments, and the other contained glucose but
did not undergo any treatment.
3.3.1
LYSOZYME
Stock solution: Lysozyme (3.4303 g ± 0.0001 g) and D-glucose (0.7700 g ±
0.0001 g) were dissolved in water (5.0 mL), to give a protein to sugar m olar ratio o f 1:18
(approximately 1:3 ratio o f lysine residues to carbonyl groups o f the glucose) for the
glucose-lysozyme mixture. Lysozym e (3.4015 g ± 0.0001 g) dissolved in water (5.0 mL)
was used as controls that underw ent treatments but did not contain any D-glucose.
Control samples o f the lysozyme that did not undergo any treatm ent were
prepared separately. Lysozyme (1.0002 g ± 0.0001 g) and D-glucose (0.2285 g ± 0.0001
g) were added to 10.0 mL o f water. They were placed at -20°C in a freezer upon
preparation for future analysis.
20
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A second set o f lysozyme samples with the addition o f sodium azide were
prepared for the incubation treatment, Lysozyme (0.9983 g ± 0.0001 g), D-glucose
(0.2297 g ± 0.0001 g) and sodium azide (0.0016 g ± 0.0001 g) were dissolved in 10.0 mL
o f water. Controls without D-glucose that contained lysozyme (1.0228 g ± 0.0001 g) and
sodium azide (0.0016 g ± 0.0001 g) were dissolved in 10.0 mL o f water.
3.3.2
BSA
Stock solution: BSA (5.0430 g ± 0.0001 g) and D-glucose (2.5180 g ± 0.0001 g)
were dissolved in water (19.0 mL), to give a protein to sugar m olar ratio o f 1:183
(approximately 1:3 ratio o f lysine residues to carbonyl groups o f the glucose) for the
BSA-glucose mixture. BSA (5.0100 g ± 0.0001 g) dissolved in w ater (19.0 mL) was used
as controls o f the experiments that underwent treatm ents but without the presence o f Dglucose.
Control samples o f BSA that did not undergo any treatm ent were prepared
separately. BSA (1.0004 g ± 0.0001 g) and D-glucose (0.5013 g ± 0.0001 g) was added to
10.8 mL o f water. They were placed at -20°C in a freezer upon preparation for future
analysis.
3.4
O V EN IN C U B A T IO N T R E A T M E N T
Lysozyme (0.18 mL), BSA (0.575 mL) samples with D-glucose and their
corresponding control samples were placed in centrifuge tubes (2.0 mL) with caps closed
so as to maintain the humidity within and prevent any oxygen from entering the sample.
All samples were placed in the Isotemp® Vacuum oven that had been preset to 30°C for
21
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
an hour to achieve a stable temperature. The incubation periods were 6, 8, 10, 12 days.
Each o f the triplicate samples was rem oved from the oven upon reaching the incubation
time and w as placed at -20°C in a freezer for later analyses.
3.5
HH P TREATMENT
Lysozym e-containing samples (0.18 mL) and BSA-containm g samples (0.575 mL)
were placed in plastic bags and heat sealed with oxygen removed, totaling up to 9 bags
for each protein (triplicates for each o f the processing time: 8 hours, 24 hours and 48
hours). The same procedure was repeated for the control samples o f lysozyme and BSA.
The above samples were placed in a bigger plastic bag filled with water and was
submerged into the pressure chamber. Pressure was attained at slightly above the desired
processing pressure prior to submerging the samples into the chamber so as to allow for
the pressure drop during processing, which can account for 1 kPsi. In our experiments,
the desired processing pressure was 400 M Pa (58 kPsi), and the preset pressure was 407
M Pa (59 kPsi). Temperature o f the cham ber (30°C) was m onitored by thermocouples
installed within the cham ber and was regulated by running hot or cold water in the jacket
around the chamber.
The three processing conditions (8 hours, 24 hours, 48 hours) were perform ed
separately, and upon reaching the processing time, the pressure o f the chamber was
released, with the rem oval o f the samples from the cham ber and stored at -20°C in a
freezer for later analyses.
22
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
3.6
F O C U S E D M IC R O W A V E IR R A D IA T IO N T R E A T M E N T
3.6.1
LY SOZY M E
Prelim inary trials starting with the original concentration o f protein and glucose
were perform ed to determine a reasonable irradiation time range. Gels formed with dark
brow ning w ere observed as the stock solutions were being m icrowaved for 1 minute at
50°C w ith varying power, therefore dilutions o f the stock solutions were done. The final
concentration used was diluted 1.6 times w ith water. The first sign o f visible browning in
the trial was seen at the 60-minute m icrowave samples. Therefore, the m icrowave time
for the samples at 50°C with varying pow er started from 10 m inutes to 60 m inutes, with a
10-minute interval between samples, totaling up to 6 different tim e treatm ent in
triplicates for both the glucose-containing samples and the controls.
Diluted samples (0.08 mL) were placed in m icrocentrifuge tubes, totaling up to
18 tubes for the 6 treatments in triplicates. Each sample was placed in the 12 cm 3 quartz
reactor o f the Prolabo unit to be m icrowaved for the desired period o f time. Tem perature
was set to rem ain constant at 50°C with varying power, and the adapter was set to rotate
clockwise during the microwave process, so as to distribute the m icrowave energy
uniformly around the sample.
3.6.2
BSA
The above procedure was also used for BSA samples. Concentration o f the BSA
samples was diluted 16 times (instead o f 1.6) with water, and 0.5 m L o f the diluted
solution placed in centrifuge tube was subjected to m icrowave treatment. Samples upon
23
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
m icrow aving for the desired processing time were stored at -20°C in a freezer for iater
analyses.
3.7
ANALYSES OF THE T R E A T E D SAMPLES
Treated samples were subjected to different dilutions prior to the analyses, SDS
(12%) w as added to the samples that gelled upon processing to aid in solubilizing the
gels for analyses. See Table 3.1 to 3.3 for the dilutions and the amount o f SDS added (if
necessary) to the treated glucose-containing samples and the controls o f lysozyme and
BSA. SDS treated samples were vortexed and centrifuged to separate the solubilized
protein fraction upon the addition o f SDS for analyses.
3.7.1
LOWRY TEST
The amount o f soluble protein in the treated samples was detected by the Lowry
m ethod (Lowry et al., 1951). Reagent A was prepared by dissolving NaaCCb (100 g) in 1
L o f NaOH (0.5 N); reagent B was prepared by dissolving CuSCL (10 g) in 1L o f water;
reagent C was prepared by dissolving sodium tartrate (20 g) in 1 L o f water. M ixing o f
the reagents A, B and C was in the ratio o f 20:1:1 in an Erlenm eyer flask (solution D).
Diluted samples (30 to 80 pL, see Table 3.1 to 3.3 for amounts o f individual samples
used) were introduced into test tubes with water added to a total volume o f 1 mL. Pure
water (1 mL) was used as blank. Solution D (1 mL) was added to the samples while
vortexing and let to stand at room tem perature for 15 m inutes. A t the end o f this time
period, 3 mL o f reagent E (10% v/v o f Folin & Ciocalteu's Phenol reagent) was forcibly
pipetted to the incubated solution while vortexing. The samples were then incubated for
45 minutes at room tem perature and m easurem ents were carried out on a Beckm an
24
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
spectrophotom eter using the wavelength at 540 nm.
Three readings at different time
intervals w ere taken and their average was used in the calculations.
3.7.2
F L U O R E S C A M IN E ASSAY
T he fluorescamine assay (Yaylavan et a l , 1992) was used to determine the free
unreacted y-amino groups in the samples. Between 30 to 80 pL (see Table 3.1 to 3.3 for
amounts o f individual samples used) samples were used for the analysis. An appropriate
amount o f borate buffer (pH 8.5, 0.2 M ) was added to each o f the samples to bring the
volume to 4 mL. Fluorescamine reagent (1 mL, 15 mg in 100 mL o f acetone) was rapidly
added, accom panied with continuous vortexing for 5 minutes. Borate buffer (4 mL)
containing no protein was used as the blank. All m easurem ents were carried out at room
tem perature on a Kontron spectrofluorom eter at excitation and em ission wavelengths o f
390 and 475 nm respectively. The instrument was calibrated using 0.01 g o f quinine
sulfate in 250 mL o f 0.1 N o f H 2SO 4. Due to the fluctuations o f the readings upon
placing the solutions into the spectrofluorometer, three readings were taken, at 1, 3 and 5
minutes after the 5 m inutes o f vortexing upon addition o f fluorescamine reagent.
Curve fitting equations were generated by Microsoft® Excel (2002) software
package.
25
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Table 3.1
Dilutions of the treated samples for analysis
Dilution
factor
C I-0
C l- 6
C l- 8
C l - 12
Cf
HHP
MW
34.3
441
441
462
427.5
34.3
676
160
BSA
sample
Dilution
factor
CI-0
C l- 6
C l- 8
C l - 12
34.3
105
105
105.
o
l
U
Lysozym e
sample
Volume
used for test
(P-L)
30
50
50
50
50
30
80
30
Lysozyme
control
Dilution
factor
C l-6
C l- 8
C l-1 0
C l-1 2
Cf
HHP
MW
462
441
441
441
34.3
676
160
Volume
used for test
(pL)
30
30
30
30
BSA
control
Dilution
factor
C l- 6
C l- 8
C l-1 0
C l - 12
34.3
105
105
105
Volume
used for test
(pL)
50
50
50
50
30
80
30
Volume
used for test
(pL)
30
30
30
30
Table 3.1 shows the dilutions done and the am ount o f the diluted samples used for tests.
‘C F refers to samples subjected to regular incubation; ‘H H P’ refers to samples subjected
to HHP; ‘M W ’ refers to samples subjected to m icrowave irradiation. The number after
the hyphen o f the Cl samples refers to the duration o f incubation in days that the samples
were subjected to. CIa refers to the lysozyme samples being subjected to regular
incubation that contained sodium azide. Refer to Table 3.2 and 3.3 for the dilutions done
on the high pressure and microwave samples o f BSA for analysis.
26
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Table 3.2
The amount o f gel recovered and BSA content of HHP treated samples
Am ount o f BSA
contained in 30 pL o f
the diluted solution
(mg)
............ ( g i...........
8-1
na
na
0.5860
r - 8-2
0.6221
0.1181
0.4791
8-3
0.2596
0.1999
0.0493
24-1
0.4092
0.0777
0.3151
24-2
0.3392
0.0644
0.2612
0.1808
24-3
0.0343
0.1392
48-1
0.4135
0.0785
0.3185
48-2
0.5844
0.1110
0.4501
48-3
0.3833
0.0728
0.2952
c8-l
0.6574
0.1248
0.5063
c8-2
0.5304
0.1007
0.4085
c8-3
1.1729
0.2227
0.9033
c24-l
0.4868
0.0924
0.3749
c24-2
0.6498
0.1234
0.5004
c24-3
0.6389
0.1213
0.4921
0.5947
c48-l
0.1129
0.4580
c48-2
0.6734
0.1279
0.5186
c48-3
0.8344
0.1584
0.6426
* To all samples, 6.6 mL o f water and 0.5 m L o f SDS were added.
Sample
W eight o f gel
removed from
the bag (g)
Calculated
amount o f
BSA in the gel
Table 3.2 shows the weight o f gel recovered from the HHP treatm ent, and the calculated
amount o f BSA used for the Lowry tests and the fluorescamine assays. The sample
names given designates the hours o f HHP treatm ent that the sample had been subjected
to. Sample names without "c" designate the BSA-glucose samples, and sample names
with "c" refers to the control samples. The num ber before the hyphen refers to the hours
o f HHP treatment that the sample has been subjected to, and the num ber after the hyphen
refers to the num ber o f triplicate (e.g. 8-1 is the sample that has been subjected to 8 hours
o f HHP, and it is the first o f the triplicate; c8-l is the control sample is the first o f the
triplicate that has been subjected to 8 hours o f HHP). A 30 juL o f each o f the diluted
solutions was used for Lowry test and fluorescam ine assay. See Appendix A3.1 for a
sample calculation o f the amount o f BSA.
27
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Table 3.3
Amount of water, SDS and the calculated BSA content in the microwave
treated sam ples
W ater
Sample
added
GiL)
10-1
10-2
10-3
20-1
20-2
20-3
30-1
30-2
30-3
40-1
40-2
40-3
50-1
50-2
50-3
60-1
60-2
60-3
9970
700
670
700
700
700
SDS
added
(mL)
0.6
0.6
0.6
0.6
0.6
0.6
10000
700
700
0.6
0.6
10000
700
700
0.6
0.6
10000
700
700
0.6
0.6
10000
700
700
0.6
0.6
Am ount o f
BSA in
30pL o f the
diluted
sample
(mg)
0.0904
0.7350
0.7524
0.7350
0.7350
0.7350
0.0956
0.7350
0.7350
0.0956
0.7350
0.7350
0.0956
0.7350
0.7350
0.0956
0.7350
0.7350
Sample
W ater
added
(pL)
SDS
added
(mL)
clOOl
c l 002
c l 003
c 2001
c 2002
c2003
c3001
c30Q2
c3003
c4001
c4002
c4QQ3
c5001
c50Q2
c5003
c6001
c6002
c6003
700
700
700
500
700
700
700
700
700
700
700
700
700
700
700
700
1400
1400
0.6
0.6
0.6
0.2
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
0.6
Amount o f
BSA in
30pL o f
the diluted
sample
(mg)
0.7302
0.7302
0.7302
1.3561
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.7302
0.4746
0.4746
Table 3.3 showing the amount o f w ater and SDS. added to the gelled m icrowave samples,
and the calculated amount o f BSA in 30 pL o f the diluted solution, which is the amount
o f the diluted solution used for Lowry tests and fluorescamine assays. The digits before
the hyphen o f the sample names refer to the m icrowave time in minutes, and the digit
after the hyphen refers to the triplicate, ‘c ’ refers to the control samples (e.g. 1001 is the
sample that has been subjected to 10 m inutes o f m icrowave irradiation, and it is the first
o f the triplicate; while c l 0-1 is the control sample that has been subjected to 10 minutes
o f microwave and is the first o f the triplicate.). See Appendix A3.1 for a sample
calculation o f the amount o f BSA.
28
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
4
RESULTS
4.1
IN T R O D U C T IO N
All the samples o f lysozyme and BSA were prepared without the addition
o f sodium azide except where noted (i.e. only one incubation set o f lysozyme was
prepared w ith the addition o f sodium azide). Samples with a ‘c ’ in front referred to
the controls for that specific treatment. For all the sample nam es used, the number in
front o f the hyphen refers to the duration o f the specific treatm ent that the sample has
been subjected to; the num ber after the hyphen refers to the num ber o f triplicate for
that treatment. For incubation samples, the num ber before the hyphen refers to the
num ber o f days being incubated; for high pressure samples, it refers to the num ber of
hours being pressurized; for m icrowave samples, it refers to the num ber o f minutes
being m icrowaved. Controls that contained D-glucose but did not undergo any
treatm ent was day 0 sample and designated as ‘O’ before the hyphen in the tables that
contained the incubation samples. The 3 readings obtained from the Low ry test will
be denoted by 1st, 2nd and 3rd in the tables, ‘o f the 9X ’ refers to the average
absorbance o f the nine readings obtained from the triplicates; while the 3 readings
obtained from the fluorescamine assay were denoted by ‘@ m in V , ‘@ m in 3 ’ and
‘@ m in 5 ’. Relative fluorescence determined from the fluorescam ine assay was
abbreviated by ‘flu’ in the tables. The corresponding standard deviation o f the
readings or calculations was designated as ‘std. dev.’ in the tables.
The data o f the Low ry tests and the fluorescamine assays were all norm alized to
0.1 mg of protein for comparison, and the standard deviations o f the triplicates were
calculated. All the results are tabulated in the tables below.
29
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Reproduced
with permission
4.2
4.2.1
RESULTS OF LYSOZYME SAMPLES
RESULTS OF LOWRY TESTS
of the copyright owner. Further reproduction
Table 4.1
Normalized absorbance and calculated protein content of incubated lysozyme samples that did not contain sodium azide.
N orm alized a b s o rb a n c e
S am ple
8
1"
0-1 ! 0.820
0-2 ; 0.890
0-3
6-1
6-2
6-3
8-1
8-2
prohibited without perm ission.
<
!
!
;
!
|
8-3 ;
10-1 »
10-2
10-3 '
12-1 ;
12-2 !
12-3 |
c6-i ;
c6-2 |
c6-3 !
c8-1 !
c8-2 ;
c8-3 '
c10-1 !
cio-2;
C10-3 |
C12-1 !
C12-2 I
C12-3 ;
0.946
0.349
0.352
0.336
0.403
0.382
0.379
0.400
0.393
0.382
0.390
0.406
0.382
0.385
0.403
0.399
0.306
0.337
0.357
0.314
0.315
0.311
0.300
0.303
0.307
2nd
0.830
0.906
0.958
0.353
0.356
0.344
0.411
0.391
0.388
0.409
0.403
0.387
0.396
0.412
0.387
0.395
0.40 i
0.38 |
0.326
0.338
0.359
0.314
0.315
0.312
0.297
0.306
0.303
.
3 rt
0.831
0.910
0.959
0.357
0.361
0.345
0.414
0.392
0.390
0.411
0.407
0.393
0.400
0.421
0.399
0.391
0.406
0.379
0.323
0.333
0.359
0.308
0.315
0.314
0.293
0.304
0.304
.
3,d
. ,
.
Protein
content(% )
C orresponding
std d e v
"
std .d ev .
2nd
0.886 0.06279 .0.898.....
0.06423
0.900
0.06440
0.895 0.05568
100
6.2243
0.346 0.00870 0.351
0.00639
0.355
0.00845
0.350 0.00790
39.16
0.88
0.388 0.01335 0.397
0.01257
0.399
0.01335
0.395 0.01240
44.11
1.39
0.00930 0.400
0.01102
0.404
0.00966
0.398 0.01022
44.52
1.14
0.392 0.01203 0.399
0.01261
0.407
0.01252
0.399 0.01243
44.63
1.39
0.396 0.00974 0.395
0.01288
0.392
0.01365
0.394 0.01071
44.07
1.20
0.333 0.02611 0.341
0.01654
0.338
0.01835
0.338 0.01829
37.74
2.04
0.313 0.00208 0.314
0.00136
0.312
0.00361
0.313 0.00227
35.01
0.25
0.303 0.00343 0.302
0.00417
0.301
0.00630
0.302 0.00431
33.76
0.48
1
0.391
std . d ev
30
std . d ev
of th e 9X
std. dev.
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.2
Normalized absorbance and the calculated protein content o f the lysozyme samples that contain glucose and 0.01%
sodium azide being subjected to regular incubation at 30°C from day 0 to day 12
N orm alized a b s o rb a n c e
S a m p le
prohibited without perm ission.
0-1
0-2
0-3
2-1
2-2
2-3
6-1
6-2
6-3
8-1
8-2
8-3
10-1
10-2
10-3
12-1
12-2
12-3
i«
i . J . .....................
:
|
|
;
!
!
1.040
1.072
0.982
1.035
1.012
1.029
1.037
0.958
1.028
1.000
1.033
0.959
1.017
0.992
1.009
0.916
1.074
0.988
2nd
1.061
1.095
1.004
1.055
1.041
1.052
1.062
0.981
1.051
1.014
1.053
0.968
1.041
1.010
1.031
0.926
1.096
1.014
3 '“
1.064
1.102
1.006
1.058
1.047
1.055
1.063
0.981
1.060
1.018
1.058
0.969
1.043
1.011
1.033
0.926
1.095
1.015
A v e ra g e norm alized a b s o rb a n c e
1*
std .d ev .
2 nd
s td .d e v .
!
3rd
s td .d e v .
of the 9X
std. dev.
P rotein
co n ten t
(%)
C orresponding
s td dev.
1.031.... 0 0 4 5 3 0
1.053
0.04573
1.057
0.04803
’1.047
004195
100
4.00529
1.026
0.01188
1.049
0.00707
1.053
0.00553
1.043
0.01497
99.58
1.42961
1.008
0.04306
1.031
0.04388
1.035
0.04637
1.025
0.04056
97.82
3.87239
0.998
0.03689
1.012
0.04287
1.015
0.
1.008
0.03695
96.26
3.52847
1.006
0.01290
1.027
0.01593
1.029
0.01607
1.021
0.01710
97.47
1.63299
0.993
0.07913
1.012
0.08509
1.012
0.08452
1.005
0.07246
95.99
6.91895
31
Reproduced
with permission
of the copyright owner. Further reproduction
T able 4.3
Normalized absorbance and the calculated protein content o f the control samples o f lysozym e with 0.01% sodium azide
being subjected to regular incubation from day 0 to day 12
N orm alized a b s o rb a n c e
S am ple
s 18
2 nd
A v erag e norm alized a b s o rb a n c e
3 rd
c 0 -1
1 .0 7 5
1 .0 8 2
1 .0 8 7
cO-2
1 .1 5 5
1 .1 7 2
1 .1 7 3
c O -3
1 .0 5 2
1 .0 6 0
1 .0 6 1
c2-1
1 .0 2 6
1 .0 3 5
1 .0 3 8
c 2 -2
0 .9 1 0
0 .9 1 3
0 .9 1 3
c2-3
1 .0 0 7
1 .0 2 0
1 .0 2 0
prohibited without perm ission.
c 6 -1
1 .0 0 7
1 .0 2 1
1 .0 2 6
c6-2
0 .9 4 7
0 .9 5 4
0 .9 5 6
c 6 -3
1 .0 9 5
1 .1 0 4
1 .1 0 6
c 8 -1
1 .0 8 9
1 .0 9 7
1 .0 9 8
c 8 -2
1 .1 1 8
1 .1 2 2
1 .1 2 6
c 8 -3
1 .1 0 0
1 .1 0 7
1 .1 0 7
C 1 0 -1
1 .0 6 2
1 .0 7 5
1 .0 7 4
C 1 0 -2
1 .0 1 2
1 .0 2 2
1 .0 2 2
C 1 0 -3
1 .0 5 4
1 .0 6 5
1 .0 6 6
C12-1
C12-2
1 .1 2 0
1 .1 3 1
1 .1 3 2
0 .9 9 8
1 .0 0 2
1 .0 0 2
C 1 2 -3
0 .9 9 2
1 .0 0 8
1 .0 1 1
2nd
s td .d e v .
!
3 rd
s td .d e v .
of th e 9X
std . dev.
Protein
co n te n t
(% )
C o rresp o n d in g
s td dev.
1 .0 9 4
0 .0 5 4 3 3
1 .1 0 5
0 .0 5 9 2 1
1 .1 0 7
0 .0 5 8 5 7
1 .1 0 2
0 .0 5 0 0 8
1 0 5 .2 3
4 .7 8 1 6 2
0 .9 8 1
0 .0 6 1 9 2
0 .9 8 9
0 .0 6 6 7 4
0 .9 9 0
0 .0 6 7 5 6
0 .9 8 7
0 .0 5 6 8 5
9 4 .2 3
5 .4 2 8 3 9
1 .0 1 6
0 .0 7 4 6 5
1 .0 2 6
0 .0 7 4 9 2
1 .0 2 9
0 .0 7 4 8 5
1 .0 2 4
0 .0 6 5 0 5
9 7 .7 8
6 .2 1 1 2 1
1 .1 0 2
0 .0 1 4 2 8
1 .1 0 9
0 .0 1 3 0 3
1 .1 1 0
0 .0 1 4 3 9
1 .1 0 7
0 .0 1 2 5 8
1 0 5 .7 1
1 .2 0 0 7 7
1 .0 4 3
0 .0 2 7 1 8
1 .0 5 4
0 .0 2 8 0 5
1 .0 5 4
0 .0 2 7 8 5
1 .0 5 0
0 .0 2 4 6 5
1 0 0 .2 9
2 .3 5 3 5 1
1 .0 3 6
0 .0 7 2 4 8
1 .0 4 7
0 .0 7 2 4 8
1 .0 4 8
0 .0 7 2 5 4
1 .0 4 4
0 .0 6 3 0 4
9 9 .6 8
6 .0 1 9 0 9
32
Reproduced
with permission
of the copyright owner. Further reproduction
T able 4.4
N orm alized absorbance and the calculated protein content o f the lysozym e samples being subjected to HHP treatment
from 8 hours to 48 hours at 400 MPa
Norm alized a b s o rb a n c e
S a m p le
8 -1
prohibited without perm ission.
8-2
8-3
24-1
24-2
24-3
48-1
48-2
48-3
c 8-1
c 8-2
c 8-3
c 24-1
c 24-2
c 24-3
c 48-1
c 48-2
c 48-3
1*
0.759
0.689
0.693
0.700
0.702
0.687
0.725
' 0.700
0.685
0.774
0.766
0.750
0.807
0.766
0.752
0.783
0.748
0.748
2 nd
0.760
0.698
0.707
0.715
0.711
0.702
0.728
0.706
0.691
0.787
0.778
0.763
0.820
0.780
0.765
0.792
0.757
0.761
A v e ra g e norm alized a b s o rb a n c e
3'd
0.763
0.699
0.709
0.716
0.712
0.704
0.728
0.707
0.691
0.788
0.778
0.763
0.824
0.787
0.779
0.796
0.758
0.766
Protein
co n ten t {%)
C o rresp o n d in g
s td dev.
1"
std.dev.
2 nd
std . dev.
3 rd
std . dev.
of th e 9X
0.713
0.03936
0.722
0.03363
0.724
0.03409
0.720
0.03136
80.40
3.50344
0.696
0.00789
0.709
0.00666
0.711
0.00594
0.705
0.00900
78.82
1.00518
0.703
0.02027
0.708
0.01830
0.709
0.01822
0.707
0.01660
78.96
1.85507
0.763
0.01198
0.776
0.01186
0.776
0.01247
0.772
0.01230
86.25
1.37422
0.775
0.02847
0.788
0.02828
0.797
0.02372
0.786
0.02518
87.88
2.81321
0.759
0.02038
0.770
0.01934
0.773
0.02001
0.768
0.01836
85.77
2.05099
std . dev.
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.5
Normalized absorbance and the calculated protein content o f the lysozyme samples that contained glucose being
subjected to microwave irradiation from 10 minutes to 60 minutes
N orm alized a b s o rb a n c e
A v erag e norm alized a b s o rb a n c e
Protein
co n te n t
S a m g ie
1 001
0 .4 6 4 6
0 .4 7 9 9
0 .4 8 2 7
1002
0 .4 8 9 7
0 .5 0 9 3
0 .5 1 3 2
1003
0 .4 5 9 6
0 .4 8 1 8
0 .4 8 7 1
2001
0.5122
0 .5 3 3 6
0 .5 3 9 0
2002
0 .5 8 1 3
0 .5 9 4 4
0 .5 9 8 3
2003
0 .6 0 7 5
0 .6 3 3 7
0 .6 3 8 9
3001
0 .5 6 8 5
0 .5 9 2 6
0 .6 0 0 7
3002
0 .6 2 9 8
0 .6 5 5 9
0 .6 6 2 5
prohibited without perm ission.
3003
0 .5 7 7 4
0 .5 8 9 2
0 .5 9 0 5
4001
0 .5 2 1 6
0 .5 5 5 1
0 .5 6 4 5
4002
0 .4 7 5 3
0 .5 1 0 6
0 .5 2 3 7
4003
0 .5 5 9 1
0 .5 8 3 9
0 .5 9 0 5
5 001
0 .5 1 1 9
0 .5 3 6 8
0 .5 4 3 4
5002
0 .5 6 5 6
0 .5 9 0 5
0 .5 9 7 0
5003
0 .5 6 8 2
0 .5 9 4 4
0 .6 0 1 0
6001
0 .5 8 0 5
0 .6 0 4 7
0 .6 1 5 4
6002
0 .5 3 6 8
0 .5 6 1 7
0 .5 7 3 5
6003
0 .5 7 7 4
0 .5 9 7 0
0 .6 0 3 6
C orresp o n d in g
std dev.
std. dev.
of th e 9X
0 .4 9 4 3
0 .0 1 6 5 1
0 .4 8 5 3
0 .0 1 7 7 4
5 4 .2 3
1 .9 8 1 7 1
0 .0 5 0 4 2
0 .5 9 2 1
0 .0 5 0 2 7
0 .5 8 2 1
0 .0 4 4 7 9
6 5 .0 4
5 .0 0 4 9 6
0 .6 1 2 6
0 .0 3 7 6 0
0 .6 1 7 9
0 .0 3 8 9 7
0 .6 0 7 4
0 .0 3 3 8 9
6 7 .8 7
3 .7 8 6 9 9
0 .0 4 1 9 7
0 .5 4 9 9
0 .0 3 6 9 3
0 .5 5 9 6
0 .0 3 3 6 6
0 .5 4 2 7
0 .0 3 7 5 2
6 0 .6 4
4 .1 9 2 2 0
0.5486
0 .0 3 1 7 8
0 .5 7 3 9
0 .0 3 2 1 9
0 .5 8 0 4
0 .0 3 2 1 9
0 .5 6 7 6
0 .0 3 1 3 5
6 3 .4 2
3 .5 0 2 6 1
0 .5 6 4 9
0 .0 2 4 3 9
0 .5 8 7 8
0 .0 2 2 9 4
0 .5 9 7 5
0 .0 2 1 6 2
0 .5 8 3 4
0 .0 2 4 6 4
6 5 .1 8
2 .7 5 2 7 8
1s*
std .d ev .
0 .4 7 1 3
0.01612
0 .4 9 0 4
0 .0 1 6 4 4
0 .5 6 7 0
0 .0 4 9 2 6
0 .5 8 7 2
0 .5 9 1 9
0 .0 3 3 1 1
0 .5 1 8 6
34
std. dev.
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.6
N orm alized absorbance and the calculated protein content o f the control samples o f lysozyme being subjected to
microwave irradiation from 10 minutes to 60 minutes
N orm alized a b s o rb a n c e
S a m p le
prohibited without perm ission.
C1001
C1002
C1003
C2001
C2002
C2003
C3001
C3002
C3003
C4001
C4002
C4003
C5001
C5002
C5003
C6001
C6002
C6003
1”
0.7082
0.6760
0.6800
0.6044
0.7381
0.7183
0.5868
0.5994
0.6166
0.6152
0.3776
0.6628
0.6153
0.7038
0.6047
0.8126
0.7117
0.6074
A v erag e norm alized a b s o rb a n c e
Protein
co n te n t
2 nd
3fd
0.7291
0.6945
0.7130
0.6166
0.7500
0.7354
0.6206
0.6166
0.6325
0.6422
0.3895
0.6853
0.6377
0.7315
0.6166
0.8491
0.7394
0.6192
0.7366
0.7024
0.7236
0.6206
0.7552
0.7407
0.6341
0.6232
0.6391
0.6517
0.3895
0.6985
0.6523
0.7354
0.6245
0.8640
0.7526
0.6258
std .d ev .
std . d e v .
> 3
std . dev.
of th e 9X
std. dev.
%
C o rresp o n d in g
s td dev.
0.6881
0.01753
0.7122
0.01733
0.7209
0.01726
0.7070
0.02105
79.00
2.35221
0.6869
0.07215
0.7007
0.07319
0.7055
0.07389
0.6977
0.06384
77.96
7.13305
0.6010
0.01496
0.6232
0.00824
0.6321
0.00811
0.6188
0.01682
69.14
1.87881
0.5519
0.15278
0.5723
0.15979
0.5799
0.16653
0.5680
0.13896
63.47
15.52617
0.6413
0.05438
0.6619
0.06114
0.6707
0.05772
0.6580
0.05176
73.52
5.78276
0.7106
0.10263
0.7359
0.11497
0.7475
0.11915
0.7313
0.09877
81.71
11.03548
35
Reproduced
with permission
4.2.2 R ESULTS OF FLU O R ESC AM IN E ASSA Y S
Table 4.7
The calculated extent of glycation or denaturation o f the lysozyme incubated samples without sodium azide from day 0 to day 12
of the copyright owner. Further reproduction
t
N orm alized relative flu o re sc a n c e (Flu.)
@rnm1
S am ple
t
0-1
[
112.4
120.8
122.7
0-2
i
i
106.3
113.5
114.5
117.3
125.5
124.2
0-3
@ m in 3
@ m in 5
6-1
'
20.3
21.2
21.2
6-2
|
22.6
23.4
24.1
6-3
'
20.7
21.2
21.2
8-1
I
25.3
26.9
26.8
8-2
J
27.1
27.9
27.6
8-3
1i
i
25.7
26.1
26.3
10-1
22.5
23.4
23.1
10-2
!
19.3
19.8
19.4
prohibited without perm ission.
10-3
]
25.8
26.8
26.5
12-1
'
24.5
24.4
24.5
12-2
i
23.4
24.1
24.1
21.6
22.4
22.3
12-3
[
c6-1
|
43.2
45.0
44.3
c6-2
'
41.5
43.7
42.9
c6-3
!
38.7
40.6
39.5
c8-1
|
37.0
38.2
37.9
c8-2
1g
36.7
38.3
37.1
c8-3
i
34.8
36.3
36.0
C10-1
'
34.3
35.1
34.8
C10-2 j
34.0
35.1
34.8
C10-3 '
32.3
32.5
31.8
C12-1
!
27.4
29.1
28.0
C12-2
27.2
27.7
27.3
C12-3 !
27.4
28.0
27.2
A verage
flu of
triplicate
Flu
co n ten t
Amt
protein
from Lowry
test (mg)
A v erag e
#of
E x p ected
flu o re sc e n c e
from Lowry
D ifference
in
flu o re sc e n c e
# tysyl g ro u p s got
G lycated or
de n a tu re d
reacted
E xtent of
glycation or
d en atu ratio n
C orresponding
(%)
std dev.
std . dev.
%
% drop
in flu
117.5
6.34991
100.00
-
0.1000
117.500
0
0
0
0
6.34991
21.8
1.28196
18.55
81.45
0.0392
46.000
24.214
8.65E-09
1.24
20.61
1.21261
26.6
0.85255
22.67
77.33
0.0441
51.815
25.187
8.99E-09
1.29
21.44
0.68633
23.0
2.98966
19.55
80.45
0.0445
52.297
29.331
1.05E-08
1.50
24.96
3.24960
23.5
1.10622
20.00
80.00
0.0446
52.426
28.923
1.03E-08
1.48
24.62
1.15918
42.2
2.20501
35.89
64.11
0.0441
51.768
9.607
3.43E-09
0.49
8.18
0.42763
36.9
1.14588
31.45
68.55
0.0377
44.332
7.394
2.64E-09
0.38
6.29
0.19520
33.8
1.29109
28.81
71.19
0.0350
41.125
7.280
2.60E-09
0.37
6.20
0.23636
27.7
0.60436
23.57
76.43
0.0338
39.657
11.968
4.27E-09
0.61
10.19
0.22233
tysyl
g ro u p s
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.8
The calculated extent o f glycation o f lysozym e samples that contained glucose and 0.01% sodium azide being
subjected to regular incubation from day 0 to day 12
Norm alized relative flu o re sc e n c e
(Flu)
S a m p le
prohibited without perm ission.
0-2
0-3
2-1
2-2
2-3
6-1
6-2
6-3
8-1
8-2
8-3
10-1
10-2
10-3
12-1
1 2 -2
12-3
@ m in 1
67.0
76.0
68.7
74.5
71.8
53.9
67.1
66.9
67.3
71.0
66.5
60.6
69.4
6 6 .2
64.4
53.2
73.5
66.7
® m in 3
70.8
82.1
73.2
78.4
76.1
56.7
70.8
70.5
70.4
76.1
68.7
63.4
72.6
70.7
67.8
56.4
78.2
70.7
@ m in 5
70.6
82.1
71.8
78.3
76.3
55.6
70.1
69.5
70.4
74.9
68.5
63.1
71.9
68.4
67.2
56.0
78.3
68.8
A v erag e
flu
of
triplicate
std . dev.
Flu
C o n ten t
%
73.6
100.00
69.1
93.88
69.2
% d rop
in flu
Amt protein
from Lowry
te s t
(m g)
E x p e c te d
D ifference
flu o re sc e n c e
in
from Low r y ____ f lu o r e s c e n c e ^
# lysyl g ro u p s
th at got
glycated
A v e ra g e #
of re a c te d
iysy!
g ro u p s
Extent of
glycation
C orresponding
....
0.1000
73.600
6.12
0.0996
73.289
4.211
2.40E-09
0.34
5.72
0.86550
94.06
5.94
0.0978
71.965
2.752
1.57E-09
0.2;
3.74
0.08814
68.1
92.53
7.47
0.0963
70.861
2.775
1.58E-09
0.23
3.77
0.29477
68.8
93.44
6.56
0.0975
71.744
2.987
1.70E-09
0.24
4.06
0.15848
66.9
90.87
9.13
0.0960
70.640
3.774
2.15E-09
0.31
5.13
0.73602
37
5.44594
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.9
The calculated extent o f denaturation o f control samples o f lysozym e that contained 0.01% sodium azide when being
subjected to regular incubation from day 0 to day 12
N orm alized relative flu o rescen ce
(Flu)
Am t protein
from Lowry
te s t
prohibited without perm ission.
@mtrt 5
A v erag e
flu, of
triplicate
7 3 .6
7 3 .3
7 3 .9
3 .9 6 4 4 7
1 0 0 .4 0
-0 .4 0
0 .1 0 5 2
7 7 .4 1 0
3 .5 3 4
7 4 .9
7 9 .0
8 0 .4
cQ-3
6 7 .4
7 2 .8
7 2 .7
c 2 -1
6 8 .9
7 2 .8
7 2 .6
6 9 .4
5 .4 9 5 3 5
9 4 .3 0
5 .7 0
0 .0 9 4 2
6 9 .3 1 6
c 2 -2
6 1 .6
6 3 .5
6 2 .5
c 2 -3
7 2 .7
7 6 .4
7 3 .5
c 6 -1
6 9 .7
7 3 .3
6 9 .8
7 1 .2
3 .2 7 7 8 7
9 6 .7 9
3 .2 1
0 .0 9 7 8
c 6 -2
6 6 .2
6 8 .8
6 9 .4
c 6 -3
7 2 .8
7 6 .9
7 4 .1
c 8 -1
7 2 .9
7 6 .9
7 6 .1
6 8 .6
5 .6 2 7 8 7
9 3 .1 8
6 .8 2
7 0 .1
2 .7 2 8 7 7
9 5 .2 9
6 9 .2
4 .9 0 9 7 2
9 3 .9 8
S a m p le
@ m in 1
c 0 -1
7 0 .8
c O -2
@ m in 3
c 8 -2
6 1 .0
6 3 .0
6 5 .5
c 8 -3
6 5 .4
6 7 .7
6 8 .5
c10-1
7 0 .0
7 4 .0
7 4 .1
C 1 0 -2
6 6 .0
6 9 .8
6 8 .3
dO -3
6 9 .0
7 1 .8
6 8 .1
C 1 2 -1
7 2 .4
7 5 .9
7 6 .7
C 1 2 -2
6 2 .8
6 5 .5
6 5 .1
C 1 2 -3
6 6 .0
6 8 .8
6 9 .3
std . dev.
E xp ected
flu o re sc e n c e
E xtent of
d e n a tu ra tio n
(%)
C orresponding
std dev.
2.01 E-09
0 .2 9
4 .8 0
0 .2 5 7 7 0
- 0 .0 7 1
-4.05E-11
- 0 .0 1
-0.10
-0.00765
7 1 .9 6 5
0 .7 4 6
4.25E -10
0 .0 6
1.01
0.04664
0 .1 0 5 7
7 7 .7 7 8
9 .2 1 6
5.25E-09
0 .7 5
1 2 .5 2
1 .0 2 7 7 8
4 .7 1
0 .1 0 0 3
7 3 .8 0 4
3 .6 8 5
2.10E-09
0 .3 0
5 .0 1
0 .1 9 4 8 3
6 .0 2
0 .0 9 9 7
7 3 .3 6 3
4 .2 1 2
2.40E-09
0 .3 4
5 .7 2
0 .4 0 6 2 8
% d rop
in flu
38
D ifference
in
ft lysyl g ro u p s
th at got
d e n a tu re d
A v e ra g e #
of re a c te d
lysyl g ro u p s
Flu
c o n te n t %
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.10
T he calculated extent o f glycation or denaturation o f lysozym e samples being subjected to HHP from 8 hour to 48
hours at 400 MPa
N orm alized relative
flu o re sc e n c e (Flu)
Amt protein
from Lowry
te s t
(m g)
# lysyl
g ro u p s
got
g ly cated o r
d e n a tu re d
prohibited without perm ission.
I @min1
2jmin3
@ m in5
A v erag e
flu.
of th e
triplicate
std . dev.
Flu %
% d rop
in flu %
8 -1
[ 4 6 .7
4 8 .0
4 8 .7
4 7 .3
1 .1 8 3 1 5
4 0 .2 1 3
5 9 .7 9
0 .0 8 0 4
1 1 3 .8 1 9
6 6 .5 6 9
2.38E-08
3 .4 0
5 6 .6 5
141862"
8 -2
' 4 6 .3
4 9 .0
4 7 .2
8 -3
!
4 7 .6
4 6 .3
4 6 .1
1 .3 3 4 8 4
3 9 .2 0 8
6 0 .7 9
0 .0 7 8 8
9 2 .6 1 4
4 6 .5 4 4
1.66E-08
2 .3 8
3 9 .6 1
1 .1 4 7 7 4
2 4 -3
45.5
; 45.0
« 45.1
! 43.6
4 8 -1
5 0 .8
4 9 .6
2 .1 4 9 1 5
4 2 .2 1 2
5 7 .7 9
0 .0 7 9 0
9 2 .7 7 8
4 3 .1 7 9
1.54E-08
2 .2 0
3 6 .7 5
1 .5 9 2 3 3
4 8 -2
\ 4 8 .6
5 1 .3
5 0 .3
4 8 -3
• 4 5 .6
4 8 .2
4 8 .0
c 8 -1
1 5 4 .9
5 8 .3
5 7 .4
5 6 .4
1 .8 1 2 4 7
4 7 .9 8 6
5 2 .0 1
0 .0 8 6 3
1 0 1 .3 4 4
4 4 .9 6 0
1.61E-08
2 .3 0
3 8 .2 6 ’ ’ Y .2 3 0 6 2
c 8 -2
! 5 4 .0
5 7 .5
5 7 .8
c 8 -3
a1 5 3 . 3
5 7 .0
5 7 .3
c 2 4 -1
' 5 9 .1
6 2 .2
6 1 .6
5 7 .0
3 .3 4 5 1 3
4 8 .4 9 3
5 1 .5 1
0 .0 8 7 9
1 0 3 .2 5 9
4 6 .2 8 0
1.65E-08
2 .3 6
3 9 .3 9
2 .3 1 2 3 6
4 9 .1
3 .9 2 1 0 2
4 1 .7 9 3
5 8 .2 1
0 .0 8 5 8
1 0 0 .7 8 0
5 1 .6 7 3
1.85E-08
2 .6 4
4 3 .9 8
3 .5 1 1 4 3
I
S am ple
2 4 -1
2 4 -2
4 7 .5
4 6 .2
4 7 .6
4 7 .3
4 6 .0
4 6 .4
5 2 .6
5 0 .8
c 2 4 -2
!
5 6 .1
;
54.5
53.1
5 7 .5
c 2 4 -3
5 4 .9
5 3 .9
c 4 8 -1
| 5 0 .6
5 2 .9
5 1 .6
c 4 8 -2
!
49.8
53.5
5 1 .0
c 4 8 -3
! 4 2 .6
4 5 .4
4 4 .6
39
E x p e c te d
flu o re sc e n c e
from Lowry
Difference
in
fluoresc e n ce
A v e ra g e $
of re a c te d
lysyl g ro u p s
Extent of
Glycation or
den a tu ra tio n
...(%).....
C orresponding
std d ev
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.11
The calculated extent o f glycation o f lysozym e samples that contained glucose being subjected to microwave
irradiation from 10 minutes to 60 minutes
N orm alized R elative flu o re sc e n c e
(Flu)
S am p le
prohibited without perm ission.
1001
1002
1003
2001
2002
2003
3001
3002
3003
4001
4002
4003
5001
5002
5003
6001
6002
6003
@ min1
25.9
26.3
24.1
28.6
27.6
31.9
19.8
35.0
29.7
30.2
29.6
28.8
27.8
28.0
28.5
34.5
29.5
26.2
@ m in3
27.1
27.4
25.1
29.5
29.2
30.8
20.2
36.4
31.9
32.4
31.2
30.1
28.0
28.5
29.5
35.3
31.0
27.0
@ m in5
26.7
27.0
24.6
29.6
28.5
30.5
20.0
36.3
31.6
31.2
30.1
30.4
28.4
A v erag e
flu. of
the
triplicate
Flu
co n te n t
%
%
d rop
in flu
Amt protein
from Lowry
te s t
(m g)
E x p e c te d
flu o re sc e n c e
from Lowry
D ifference
in
# tysyl
g ro u p s
got
glycated _
A v erag e #
of re a c te d
tysyi g ro u p s
E xtent of
glycation
C orresponding
s td d ev
(%1 ..............
2 6 .0
1 .1 7 2 3 4
2 2 .1 5
77.85
0 .0 5 4 2
6 3 .7 2 0
3 7 .6 9 7
1.35E-08
1 .9 2
3 2 .0 8
1 .4 4 5 3 5
2 9 .6
1 .3 2 1 2 4
2 5 .1 8
74.82
0 .0 6 5 0
7 6 .4 2 2
4 6 .8 3 6
1.67E-08
2 .3 9
3 9 .8 6
1 .7 8 0 1 0
2 9 .0
7 .0 7 6 8 6
2 4 .6 7
75.33
0 .0 6 7 9
7 9 .7 4 7
5 0 .7 5 7
1.81E-Q8
2 .5 9
4 3 .2 0
1 0 .5 4 5 2 2
3 0 .4
1 .0 5 3 2 5
2 5 .9 1
74.09
0 .0 6 0 6
7 1 .2 5 2
4 0 .8 0 7
1.46E-08
2 .0 8
3 4 .7 3
1 .2 0 1 4 4
2 8 .5
0 .5 5 4 6 2
2 4 .2 7
75.73
0 .0 6 3 4
7 4 .5 1 9
4 6 .0 0 5
1.64E-08
2 .3 5
3 9 .1 5
0 .7 6 1 5 9
3 0 .8
3 .5 9 0 8 0
2 6 .1 8
73.82
0 .0 6 5 2
7 6 .5 8 7
4 5 .8 2 1
1.64E-08
2 .3 4
3 9 .0 0
4 .5 5 1 4 2
.
28.7
29.2
35.4
30.6
27.5
40
Reproduced
with permission
Table 4.12
The calculated extent o f denaturation o f lysozym e controls being subjected to microwave irradiation from 10 minutes
to 60 minutes.
of the copyright owner. Further reproduction
N orm alized relative flu o re sc e n c e
(Flu)
S a m p ie
@rrtin 1
@ m in3
@ m in5
prohibited without perm ission.
C1001
3 7 .0
3 9 .2
3 8 .0
C1002
3 9 .0
3 9 .5
3 9 .0
C1003
3 7 .9
3 8 .0
3 9 .0
C 2001
3 3 .3
3 3 .7
3 3 .9
C2002
4 1 .3
4 3 .2
4 1 .5
C2003
4 4 .8
4 5 .6
4 5 .7
C 3001
3 0 .2
3 1 .4
3 1 .5
C3002
2 7 .2
2 9 .8
2 9 .8
C3003
3 1 .3
32.3
3 2 .2
C 4001
2 9 .3
2 9 .6
2 8 .7
C4002
1 7 .4
18.1
1 8 .0
C4003
4 3 .0
4 3 .7
4 4 .0
C 5001
3 5 .8
3 7 .5
3 6 .8
C5002
4 2 .0
4 2 .6
4 2 .6
C5003
32.6
3 3 .9
3 3 .7
C 6001
4 8 .8
5 2 .5
5 0 .7
C6002
41.1
4 2 .8
4 2 .4
C6003
3 1 .3
3 2 .3
3 1 .4
A v erag e
flu of
the
triplicate
Flu
co n ten t
std . dev.
%
%
d rop
in flu
Ami protein
from Lowry
lest
..........<m9>
E x p ected
f lu o re sc e n c e
from Lowry
Difference
in
flu o re sc e n c e
# lysyl
g ro u p s
got
d e n a tu re d
A v e ra g e #
re a c te d
lysyl g ro u p s
E xtent of
de n a tu ra tio n
(%)
C orresponding
s td d e v
3 8 . 5 ...... o ] b 6 7 4 3 "
3 2 .7 6
67.24
0 .0 7 9 0
9 2 .8 2 5
5 4 .3 2 6
1.94E-08
2 .7 7
4 6 .2 4
0 .9 6 9 6 9
4 0 .3
5 .2 5 7 7 2
3 4 .3 1
65.69
0 .0 7 8 0
9 1 .6 0 3
5 1 .2 8 9
1.83E-08
2 .6 2
4 3 .6 5
5 .6 9 2 8 3
3 0 .6
1 .5 9 9 4 2
2 6 .0 8
73.92
0 .0 6 9 1
8 1 .2 4 0
5 0 .5 9 9
1.81E-08
2 .5 8
4 3 .0 6
2 .2 4 7 8 9
3 0 .2
1 1 .1 8 0 3 0
2 5 .7 0
74.30
0 .0 6 3 5
7 4 .5 7 7
4 4 .3 7 7
1.58E-08
2 .2 7
3 7 .7 7
1 3 .9 8 1 4 6
3 7 .5
3 .9 9 6 9 2
3 1 .9 3
68.07
0 .0 7 3 5
86386
4 8 .8 7 3
1.75E-08
2 .5 0
4 1 .5 9
4 .4 3 1 7 7
4 1 .5
8 .2 9 4 3 3
3 5 .3 0
64.70
0 .0 8 1 7
' 9 6 .0 0 9
5 4 .5 3 5
1.95E-08
2 .7 8
4 6 .4 1
9 .2 8 1 9 8
41
Reproduced
with permission
of the copyright owner. Further reproduction
RESULTS OF B SA SAM PLES
4.3.1 R ESULTS OF LO W R Y TESTS
Table 4.13
Normalized absorbance and calculated protein content o f B SA samples subjected to regular incubation
4.3
N orm alized a b s o rb a n c e
S am ple
0 -1
'
0 .5 7 8
0 .5 9 3
0 .5 9 5
0 -2
i
0 .5 6 0
0 .5 8 0
0 .5 8 3
0 -3
!
0 .5 6 8
0 .5 8 3
0 .5 7 8
6 -1
;
0 .5 4 5
0 .5 5 9
0 .5 6 3
6 -2
1
0 .6 0 2
0 .6 0 2
0 .6 1 5
I
I
6 -3
.
0 .5 7 8
0 .5 8 9
0 .5 9 3
8 -1
|
0 .5 8 0
0 .5 9 1
0 .5 9 1
8-2
;
0 .5 4 5
0 .5 5 5
0 .5 6 3
0 .6 1 5
8 -3
>
0 .5 9 9
0 .6 1 0
1 0 -1
!
0 .5 1 9
0 .5 3 4
0 .5 3 4
1 0 -2
;
0 .5 8 1
0 .5 9 2
0 .5 9 3
1 0 -3
prohibited without perm ission.
1
0 .5 8 5
0 .5 9 2
0 .5 9 1
1 2 -1
i
0 .5 8 4
0 .5 8 9
0 .5 8 8
1 2 -2
|
0 .5 5 8
0 .5 6 3
0 .5 6 9
12-3
;
0 .4 7 4
0 .4 8 4
0 .4 9 2
c 6 -1
A v erag e norm alized a b s o rb a n c e
1
1
*
0 .5 0 6
0 .5 2 4
0 .5 1 7
c 6 -2
.
0 .3 7 6
0 .3 7 6
0 .3 7 6
c 6 -3
!
0 .3 9 1
0 .3 9 4
0 .3 9 7
c 8 -1
;
0 .5 3 1
0 .5 3 8
0 .5 4 1
c 8 -2
«
0 .5 4 6
0 .5 5 2
0 .5 5 2
0 .3 9 1
0 .3 9 0
;
c 8 -3
!
0 .3 8 3
c 1 0 -1
;
0 .6 1 3
0 .6 1 0
0 .6 1 0
C 1 0 -2
«
0 .5 3 4
0 .5 4 2
0 .5 4 4
C 1 0 -3
!
0 .3 8 2
0 .3 8 7
0 .3 8 7
C 1 2 -1
|
0 .5 8 2
0 .5 8 8
0 .5 8 6
C 1 2 -2
;
0 .5 3 3
0 .5 3 8
0 .5 3 9
c 1 2 -3
i
0 .3 7 9
0 .3 8 3
0 .3 7 2
Protein
std . dev.
std . dev.
std . dev.
o f th e 9X
C orresp o n d in g
0 .5 6 8
0 .0 0 9 0 4
0 .5 8 6
0 .0 0 6 8 1
0 .5 8 5
0 .0 0 8 5 7
0 .5 8 0
0 .0 1 1 0 5
1 0 0 .0 0
1 .9 0 6 8 7
0 .5 7 5
0 .0 2 8 3 0
0 .5 8 3
0 .0 2 1 9 1
0 .5 9 1
0 .0 2 6 2 1
0 .5 8 3
0 .0 2 3 1 8
1 0 0 .5 7
3 .9 9 8 5 4
0 .5 7 5
0 .0 2 7 1 4
0 .5 8 5
0 .0 2 7 8 8
0 .5 9 0
0 .0 2 6 1 1
0 .5 8 3
0 .0 2 4 3 7
1 0 0 .6 0
4 .2 0 3 8 8
0 .5 6 2
0 .0 3 6 9 3
0 .5 7 3
0 .0 3 3 3 1
0 .5 7 3
0 .0 3 3 3 4
0 .5 6 9
0 .0 3 0 4 4
9 8 .1 7
5 .2 5 0 3 6
0 .5 3 8
0 .0 5 7 4 1
0 .5 4 5
0 .0 5 5 1 0
0 .5 4 9
0 .0 5 0 8 8
0 .5 4 4
0 .0 4 7 4 7
9 3 .9 1
8 .1 8 8 3 4
0 .4 2 5
0 .0 7 1 0 8
0 .4 3 2
0 .0 8 0 7 6
0 .4 3 0
0 .0 7 6 1 7
0 .4 2 9
0 .0 6 5 9 9
7 3 .9 6
1 1 .3 8 2 2 4
0 .4 8 7
0 .0 9 0 1 6
0 .4 9 4
0 .0 8 8 9 1
0 .4 9 4
0 .0 9 0 4 1
0 .4 9 2
0 .0 7 7 8 7
8 4 .8 0
1 3 .4 3 3 0 0
0 .5 0 9
0 .1 1 7 4 1
0 .5 1 3
0 .1 1 4 1 5
0 .5 1 4
0 .1 1 4 3 3
0 .5 1 2
0 .0 9 9 8 8
8 8 .3 3
1 7 .2 2 8 4 2
0 .4 9 8
0 .1 0 5 9 8
0 .5 0 3
0 .1 0 6 7 5
0 .4 9 9
0 .1 1 2 6 8
0 .5 0 0
0 .0 9 4 0 0
8 6 .2 6
1 6 .2 1 4 9 9
42
Reproduced
with permission
of the copyright owner. Further reproduction
T able 4.14
Normalized absorbance and the calculated protein content o f BSA samples that were being subjected to H H P from 8
hours to 48 hours at 400 MPa
N orm alized a b s o rb a n c e
S a m p le
prohibited without perm ission.
8-1
8-2
8-3
24-1
24-2
24-3
48-1
48-2
48-3
c8-1
c8-2
c8-3
C24-1
C24-2
C24-3
C48-1
C48-2
C48-3
1M
0.179
0.053
0.283
0.177
0.210
0.214
0.122
0.121
0.163
0.050
0.091
0.041
0.063
0.064
0.086
0.028
0.040
0.040
2nd
0.190
0.058
0.307
0.190
0.225
0.233
0.132
0.132
0.173
0.052
0.096
0.043
0.066
0.068
0.091
0.029
0.041
0.042
A v erag e norm al z e d a b s o rb a n c e
3,d
0.191
0.060
0.318
0.196
0.231
0.237
0.136
0.134
0.179
0.053
0.098
0.044
0.068
0.070
0.093
0.029
0.043
0.043
1s’
2 nd
std .d e v
3rd
std.dev.
of th e 9X
std.dev.
Protein
c o n te n t %
C orresp o n d in g
std .d e v
0 .1 7 2
0 .1 1 4 7 8
0.185
0.124505
0.189
0.12899
0.182
0.10674
31.3852
18.4027
0 .2 0 0
0 .0 2 0 2 4
0.216
0.022941
0.222
0.02197
0.213
0.02112
36.6672
3.6410
0 .1 2 2
0 .0 0 0 5 9
0.132
0.000020
0.135
0.00102
0.129
0.00615
22.3089
1.0609
0 .0 6 0
0 .0 2 6 8 2
0.063
0.028234
0.065
0.02904
0.063
0.02437
10.8469
4.2022
0 .0 7 1
0 .0 1 3 0 4
0.075
0.013739
0.077
0.01416
0.074
0.01211
12.8172
2.0877
0 .0 3 6
0 .0 0 7 0 8
0.037
0.007357
0.038
0.00835
0.037
0.00666
6.4300
1.1475
43
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.15
Normalized absorbance and the calculated protein content o f B SA samples containing glucose that were being
subjected to microwave irradiation from 10 minutes to 60 minutes
N orm alized a b s o rb a n c e
S a m p le
prohibited without perm ission.
1001
1002
1003
2001
2002
2003
3001
3002
3003
4001
4002
4003
5001
5002
5003
6001
6002
6003
1“
0.216
0.134
0.177
0.135
0.125
0.185
0.031
0.147
0.094
0.016
0.112
0.119
0.019
0.114
0.037
0.104
0.029
0.113
2m
0.228
0.141
0.185
0.141
0.132
0.192
0.041
0.152
0.098
0.013
0.115
0.124
0.018
0.117
0.038
0.111
0.031
0.116
A v erag e norm alized a b s o rb a n c e .
3«i
Protein
co n te n t
C orresp o n d in g
std d e v
0.03759
%
31.52
6.48064
0.154
0.02826
26.48
4.87263
0.05716
0.095
0.04939
16.38
8.51634
0.084
0.06317
0.083
0.05283
14.39
9.10912
0.05216
0.058
0.05309
0.057
0.04504
9.90
7.76573
0.04783
0.089
0.05028
0.085
0.04181
14.74
7.20785
3"*
1“
std .d ev .
2 nd
std .d e v
0.233
0.144
0.187
0.144
0.134
0.194
0.039
0.153
0.100
0.012
0.116
0.125
0.018
0.118
0.038
0.116
0.031
0.119
0.176
0.04073
0.185
0.04326
0.148
0.03214
0.155
0.091
0.05797
0.082
std .d e v .
of th e 9X
std.dev.
0.188
0.04467
0.183
0.03245
0.158
0.03224
0.097
0.05560
0.097
0.05784
0.084
0.06186
0.057
0.05074
0.058
0.082
0.04625
0.086
44
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.16
Normalized absorbance and the calculated protein content o f B SA control samples that were being subjected to
microwave irradiation from 10 minutes to 60 minutes
N orm alized a b s o rb a n c e
A v erag e norm s lized a b s o rb a n c e
Protein
co n ten t
S a m p le
G 1001
prohibited without perm ission.
C1002
c1003
C2001
C2002
C2003
C3001
C3002
C3003
C4001
C4002
C4003
C5001
C5002
C5003
C6001
C6002
C6003
C o rresp o n d in g
s td d e v
1“
2nd
3,d
1“
std .d ev .
2 nd
std .d e v
3 rd
std .d e v .
o f th e SX
std.dev.
0.012
0.016
0.058
0.027
0.029
0.048
0.041
0.017
0.051
0.015
0.015
0.020
0.011
0.009
0.018
0.018
0.020
0.019
0.012
0.017
0.060
0.028
0.030
0.050
0.042
0.018
0.053
0.016
0.015
0.021
0.012
0.010
0.019
0.018
0.021
0.020
0.012
0.016
0.061
0.028
0.030
0.051
0.043
0.018
0.054
0.016
0.015
0.020
0.011
0.01
0.019
0.018
0.021
0.020
0.028
0.02530
0.030
0.02630
0.030
0.02688
0.029
0.02267
%
5.05
0.035
0.01164
0.036
0.01217
0.036
0.01232
0.036
0.01046
6.17
1.80326
0.036
0.01751
0.038
0.01814
0.038
0.01857
0.037
0.01568
6.45
2.70273
0.017
0.00283
0.017
0.00294
0.017
0.00288
0.017
0.00251
2.93
0.43256
0.013
0.00445
0.013
0.00484
0.013
0.00531
0.013
0.00425
2.26
0.73282
0.019
0.00097
0.020
0.00146
0.020
0.00165
0.020
0.00127
3.38
0.21969
45
3.90874
Reproduced
with permission
4.3.2 RESULTS OF FLUO RESCAM INE A SSAYS
Table 4.17
The calculated extent o f glycation or denaturation o f B SA samples being subjected to incubation from 0 to 12 days
of the copyright owner. Further reproduction
N orm alized relative a b s o rb a n c e (Flu)
prohibited without perm ission.
S a m p le
@ min1
@ m in3
0-1
0-2
0-3
6-1
6-2
6-3
8-1
8-2
8-3
10-1
10-2
10-3
12-1
12-2
12-3
c6-1
c6-2
c6-3
c8-1
c8-2
c8-3
C10-1
dO -2
C10-3
C12-1
C12-2
C12-3
109.5
104.8
103.9
75.0
62.8
78.2
74.3
68.7
71.2
60.4
69.4
70.5
72.3
58.4
65.2
83.2
60.8
68.4
86.3
81.0
71.9
99.3
91.1
68.6
100.8
70.7
70.1
113.3
108.4
108.1
77.6
64.7
80.6
76.9
71.2
72.9
62.0
72.1
72.3
73.5
60.2
67.9
86.1
60.4
69.4
87.1
82.3
73.1
99.7
91.5
69.6
100.7
71.6
72.2
@ m in5
113.3
113.3
108.8
78.4
64.8
81.9
77.1
71.7
73.5
62.0
71.8
73.5
74.9
61.3
67.6
86.1
60.1
69.4
87.4
82.1
73.1
100.4
91.5
69.7
101.9
73.0
71.9
A v erag e
flu
o f th e
triplicate
std . dev.
Flu co n te n t
%
109.2
3.53051
100.00
73.8
7.52474
67.54
73.0
2.76076
68.2
% drop
in
flu
Amt protein
from Lowry
te s t
E x p ected
flu o re sc e n c e
from Lowry
Difference
in
flu o re sc e n c e
# lysyl g ro u p s
got
glycated o r
d e n a tu re d
Avg. # of
r e a c te d
lysyl
9 rouPs
ex te n t of
glycation or
den a tu ra tio n
(M.....
Corresponding
s td dev.
0.1000
109.233
32.46
0.1006
109.888
36.109
3.05529E-08
20.16
33.06
3.37152
66.87
33.13
0.1006
109.888
36.842
3.11728E-08
20.57
33.73
1.27474
5.21802
62.44
37.56
0.0982
107.267
39.059
3.30483E-08
21.81
35.76
2.73547
66.8
6.01257
61.14
38.86
0.0939
102.570
35.781
3.0275E-08
19.98
32.76
2.94887
71.6
10.89651
65.53
34.47
0.0740
80.832
9.252
7.82865E-09
5.17
8.47
1.28943
80.5
6.25278
73.69
26.31
0.0848
92.630
12.139
1.02707E-08
6.78
11.11
0.86326
86.8
13.65795
79.48
20.52
0.0883
96.453
9.632
8.14968E-09
5.38
8.82
1.38712
81.4
14.79963
74.55
25.45
0.0863
94.268
12.840
1.08641E-08
7.17
11.75
2.13640
46
3.53051
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.18
The calculated extent o f glycation or denaturation o f B SA samples that were being subjected to HHP from 8 hours to
48 hours at 400 MPa
N orm alized relative
f lu o re sc e n c e (Flu)
S am ple
prohibited without perm ission.
© m in t
@ m in3
@ m in5
8-1
8-2
8-3
24-1
24-2
24-3
48-1
48-2
48-3
13.6
17.5
21
16
14
13.8
18.4
21.3
17.4
15.5
10
7
7
c8-1
2
5
2
3
5
4
2
2
1.7
10.6
8.2
7.3
13.9
2.5
6.4
3.0
3.6
5.8
4.4
3.0
2.6
1.7
13.8
19.2
21.8
16.2
15.9
10.9
8.3
7.4
12.1
2.5
5.5
3.1
3.5
5.7
4.3
3.0
2.5
1.7
c8 -2
c8-3
C24-1
C24-2
C24-3
C48-1
C48-2
C48-3
11
std .d ev .
Flu
c o n te n t
%
% d rop
in flu
Amt
protein
from Lowry
te s t
(mcj)
17.84
3.41274
16.33
83.67
0.0314
34.299
16.458
14.25
2.80367
13.04
86.96
0.0367
40.089
25.841
2.18649E-08
14.43
7.69
0.52737
7.04
92.96
0.0223
24.359
16.670
1.41049E-08
3.74
1.59454
3.43
96.57
0.0108
11.797
8.056
4.55
0.96848
4.17
95.83
0.0128
13
2.40
0.53353
2.19
97.81
0.0064
A v erag e
Ru.
of th e
triplicate
47
E x p e c te d
flu o re sc e n c e
from Lowry
D ifference
in
flu o re sc e n c e
6.991
# tysyl g ro u p s
that got
giycated or
d e n a tu re d
A v erag e #
of re a c te d
lysyl g ro u p s
9.19
E xtent of
glycation
or
d e naturation
C orresp o n d in g
m . ..................
std dev.
15.07
2.88193
9.31
15.26
1.04673
6.81623E-09
4.50
7.37
3.14319
9.43 1
7.97963E-09
5.27
4.593
3.88663E-09
2.57
1.39251E-08
1.83731
4.21
Reproduced
with permission
Table 4.19
The calculated extent o f glycation o f B SA samples which contained glucose when being subjected to microwave
irradiation from 10 minutes to 60 minutes
of the copyright owner. Further reproduction
N orm alized relative flu o re sc e n c e
(Flu)
S am p le
prohibited without perm ission.
1001
1002
1003
2001
2002
2003
3001
3002
3003
4001
4002
4003
5001
5002
5003
6001
6002
6003
@min1
14.5
16.2
18.8
15.3
14.1
12.0
2.8
14.8
6.9
2.0
8.7
9.9
2.1
11.3
2.9
2.8
1.4
7.9
@ m in3
13.5
16.9
19.6
12.9
13.6
12.4
3.7
15.4
6.2
2.2
9.1
10.1
2.4
11.8
3.0
3.2
1.5
7.9
@rnin5
A v erag e
flu.
o f the
triplicate
10.2 ... 1624
16.8
19.8
12.7
13.22
13.4
12.5
3.7
8.35
15.5
6.1
2.2
7.03
9.1
10.1
2.3
5.63
11.9
3.0
2.8
4.12
1.5
8.1
Flu
c o n ten t
% d ro p in
...........flu
Amt protein
from Lowry
te s t
(m g)
A v erag e #
of re a c te d
lysyl
g ro u p s
E x p ected
flu o re sc e n c e
from Lowry
D ifference
in
flu o re sc e n c e
0.0316
34.518
18.277
1.55E-08
10.21
<%
>
16.733
# lysyl groups
that got
glycated
E xtent
of glycation
Corresponding
std dev.
3.12483
(%
>
14.87 .. 85T3"
1.02775
12.10
87.90
0.0265
28.947
15.731
1.33E-08
8.78
14.402
1.12000
5.35067
7.64
92.36
0.0164
17.914
9.565
8.09E-09
5.34
8.757
5.61215
3.71051
6.44
93.56
0.0144
15.730
8.699
7.36E-09
4.86
7.963
4.20255
4.52365
5.16
94.84
0.0099
10.814
5.180
4.38E-09
2.89
4.742
3.80795
2.94121
3.78
96.22
0.0147
16.057
11.933
1.01 E-08
6.66
10.924
7.78950
std .d ev .
48
3.21957
Reproduced
with permission
of the copyright owner. Further reproduction
Table 4.20
The calculated extent o f denaturation o f BSA controls when being subjected to microwave irradiation from 10 minutes
to 60 minutes
N orm alized
relative f lu o re sc e n c e (Flu)
S am p le
prohibited without perm ission.
C1001
C1002
C1003
C2001
C2002
C2003
C3001
C3002
C3003
C4001
C4002
C4003
C5001
C5002
C5003
C6001
C6002
C6003
@min1
1.2
2.8
3.3
1.5
1.5
2.8
2.9
3.4
2.0
1.5
1.5
1.4
1.1
0.7
1.6
5.8
2.1
2.1
J§£rnin3
1.2
2.7
3.4
1.6
1.6
2.8
3.0
3.5
2.0
1.5
1.6
1.5
1.2
0.7
1.6
6.2
1.9
2.3
A v erag e
flu.
@ m in5
1.3
2.7
3.4
1.6
1.6
2.8
3.0
3.6
2.1
1.6
1.6
1.5
1.2
0.7
1.6
6.4
1.9
2.4
of th e
triplicate
std .d e v .
Flu
co n te n t
{%)
% d rop in
flu
Amt protein
from Lowry
te s t
(m g)
E x p e c te d
flu o re sc e n c e
from Lowry _
D ifference
in
flu o re sc e n c e
# iysyi g ro u p s
th a t got
d e n a tu re d
A v e ra g e #
re a c te d
lysyl g ro u p s
E xtent
of
d e n a tu ra tio n
(% )
C orresp o n d in g
s td dev.
2.29
1.15831
2.10
97.90
0.0050
5.462
3.172
2.68E-09
1.77
2.904
1.46940
1.98
0.61346
1.81
98.19
0.0062
6.772
4.791
4.05E-09
2.68
4.386
1.35773
2.82
0.64830
2.58
97.42
0.0064
6.991
4.170
3.53E-09
2.33
3.817
0.87723
1.53
0.05260
1.40
98.60
0.0029
3.168
1.638
1.39E-09
0.92
1.500
0.05159
1.16
0.38125
1.07
98.93
0.0023
2.512
1.348
1.14E-09
0.75
1.234
0.40426
3.46
2.01354
3.17
96.83
0.0034
3.714
0.255
2.16E-10
0.14
0.234
0.13616
49
5
DISCUSSION
5.1
EFFECT OF HHP AND MICROWAVE IRRADIATION ON LYSOZYME
Sam ples subjected to HHP and microwave irradiation did not contain sodium
azide, ail discussion in this section is referred to systems without sodium azide, unless
otherwise specified.
5.1.1
LOWRY TEST
Figure 5.1 shows the percentage o f water soluble lysozym e rem aining after
incubation for a period o f 12 days. About 40% o f the protein content was retained when
the lysozym e-glucose m ixture was incubated for 6 days, then it increased to about 44%
and rem ained relatively constant as the incubation period increased. Lysozyme is well
known as an effective antimicrobial agent in food (Ibrahim et al., 1997; Bower et al.,
1998). Its activity involves cleavage o f glycosidic bonds in the bacterial peptidoglycan,
thereby puncturing the cell walls (Sofos et al., 1998). The big drop in the lysozyme
concentration upon incubation at 30°C suggests that the lysozyme could be acting upon
m icro-organisms that present in the solution, therefore limiting the tryptophan and
tyrosine residues o f the protein. Other possible explanation for the dramatic drop in
protein content is that the protein was undergoing internal crosslinking that involved the
tryptophan and tyrosine residues. However, this could be reversible and as the incubation
time increased, the lysozym e-glucose complex released the tryptophan and;or tyrosine
residues and could be detected by the Lowry reagent. This agrees with the reported amino
acid composition o f egg albumin with glucose. Tanaka et al. (1977) reported that the
percentage o f tyrosine o f the egg album in-glucose m ixture was com paratively lower
50
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
w hen incubated for 6 days compared with 3 and 10 day incubations. This accounts for the
lower absorbance detected by the Lowry reagent. Protein loss o f the lysozyme-glucose
m ixture w as stabilized at around 55% from day 8 onwards. On the other hand, the protein
content o f the controls continuously decreased as the duration o f incubation increased.
This suggests that lysozyme by itself m ay have undergone irreversible structural changes
upon incubation, w ith the amino acids crosslinking with each other irreversibly. In other
words, this suggests that glucose acts as a protectant or a shield to the protein in
protecting the amino acids from being m odified or further crosslink, which agrees with a
previous study (Prabhakaram and Ortwerth, 1994). In addition, the protein loss for
incubated samples was high overall. This could also be due to the oxygen catalyzed free
radical dam age or degradation o f the protein.
100
c
'35
s
with g lu c o se
80
protein controls
60
®
s3
§
40
20
0
0
2
4
8
6
10
12
Time under regular incubation (days)
Figure 5.1
Percentage o f water soluble lysozyme rem ained in the samples upon being
subjected to regular incubation at 30°C over a period o f 12 days, as compared to the
initial amount o f protein at day 0.
Comparing the above result with lysozym e samples that have been incubated in
the presence o f sodium azide (Figure 5.2), it becomes clear that m icro-organism s did play
51
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
a role in degrading the lysozyme upon prolonged exposures and limiting its availability
w ith negligible decrease in the protein content for both the glucose-containing samples
and the protein controls that contained sodium azide, in which, sodium azide is
com m only used as an effective antimicrobial agent (Bower et al., 1998).
1 00
c
£
©
©
hQ,
J»
JQ
O
m
80
60
40
—#—with glucose
—
20
pr ot ei n c o n tro ls
0
8
0
10
12
Time under regular incubation (days)
Figure 5.2
Percentage o f water soluble lysozyme rem aining in the samples (that
contained 0.01% sodium azide) upon being subjected to regular incubation at 30°C over a
period o f 12 days, as com pared to the initial am ount o f protein at day 0.
Lysozyme samples, w hen subjected to 400 M Pa o f HHP at 30°C, encounter less
damage when compared to the samples subjected to regular incubation treatment (Figure
5.3). Pressure treated glucose-containing samples exhibited a 20% drop in protein content;
while for the protein controls, there was only around a 15% drop. Lysozyme has been
reported to be pressure resistant and only shows slight changes in the structure when the
protein is subjected to 300 M Pa for 30 m inutes (Tedford et al., 1999); as well as when
lysozyme is subjected to 200 M Pa for 7 days (W ebb et al., 2000). The higher pressure
and the longer duration o f pressurization applied in our experiment could explain for the
52
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
15 to 20% loss in protein content, in which some o f the secondary and the tertiary
structures o f the protein got disrupted.
M icrowave-treated samples also encountered less damage to the protein structure
as com pared to those oven-incubated without sodium azide (Figure 5.4). Protein content
o f the m icrowave-treated glucose containing samples had a higher loss when compared
w ith that o f the controls. Around 20 to 30% o f protein was lost for the lysozyme controls;
w hile the lysozyme-glucose mixture encountered a loss o f up to 40 to 55%. The highest
drop in protein content occurred for lysozyme-glucose samples subjected to 10 -minutes
o f microwaving. However, the protein content increased slightly as the m icrowave time
increased thereafter. This could be due to the reversibility o f ARP as the protein
underw ent longer exposures to m icrowave irradiation. In the absence o f glucose, the
control lysozyme samples also showed fluctuations in the amount o f soluble protein as
the microwave time increased. In any o f the two cases for the lysozym e samples that
contained glucose and the lysozyme controls, hydrogen bonds could form betw een N-H
and C = 0 groups o f adjacent turns o f the peptide spirals, and also between phenolic OH
groups and the carbonyl groups (Haurowitz, 1963). The formation o f these crosslinks
could be reversible over time, and tryptophan and tyrosine residues could involve in the
formation o f these crosslinks, therefore, accounting for the fluctuations in the amount o f
soluble protein detected by the Lowry test at the different durations o f microwave
irradiation. However, the standard deviation o f the triplicates o f the protein controls was
large overall, suggesting that lysozyme alone when being subjected to microwave
irradiation underwent more conform ational changes than when glucose was present.
53
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
100
a.
with glucose
protein controls
0
0
10
20
30
40
50
Time under high pressure (hours)
Figure 5.3
Percentage o f soluble lysozyme rem ained in samples upon being subjected
to 400 M Pa o f HHP from 8 hours to 48 hours at 30°C, as compared to the initial amount
o f protein at day 0.
100
c
O
a.
.o
3
o
m
with glucose
protein controls
0
10
20
30
40
50
60
Microwave time (minutes)
Figure 5.4
Percentage o f soluble lysozyme rem aining in samples subjected to
microwave irradiation from 10 minutes to 60 m inutes at 50°C, as com pared to the initial
amount o f protein at day 0.
54
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Out o f the three treatments, high pressure at 30°C seems to be the process that
causes the least damage to lysozyme, with 80% o f its structural integrity protected upon
processing. M icrow ave irradiation at 50°C comes next, and regular incubation at 30°C
without the presence o f antimicrobial agent such as sodium azide causes the most damage
to the protein, up to some 55% o f the protein was lost.
5.1.2
F L U O R E S C A M IN E ASSAY
Lysine, being one o f the m ost reactive am ino acids in protein, reacts readily with
glucose to generate glycated proteins. Assaying the amount o f lysine that remains in the
solution m ixture by fluorescamine assay is therefore a way to identify the amount of
intact ARP present. Figure 5.5a shows a non-linear (polynomial) correlation between the
extent o f glycation o f the lysozyme in the lysozym e-glucose m ixture that had been
incubated at 30°C. There is a linear increase in the extent o f glycation as a function o f
incubation time reaching around 20 to 25% glycation The extent o f glycation increased as
the incubation time increased, and this agrees with a study on egg albumin with glucose
(Tanaka et al., 1977) which found around 20% glycation o f egg album in upon 10 days of
incubation at 37°C. As shown in Figure 5.5a, upon 6 days o f incubation, the extent o f
glycation has reached its m aximum capacity at 30°C, with m ost o f the exposed lysyl
groups reacted. The polynomial correlation also suggested that further incubation would
induce loss o f glycated proteins, either due to reversible reactions o f ARP or crosslinks
occurring within the protein. Average num ber o f reacted lysyl groups in the samples
calculated was only around 1.3 lysyl groups (See Table 4.7 for calculated results and
Appendix A3.2 to A3.4 for steps in calculating the extent o f glycation), and the average
55
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
num ber o f reacted lysyl groups did not increase significantly after 6 days o f incubation,
and this agrees with a previous study on glycation o f lysozyme (Yeboah et al., 2000). The
difference in the average number o f reacted lysyl groups can be accounted by the
differences in the tem perature o f the two experiments. Since tem perature has been shown
as a significant factor in promoting M aillard reaction, therefore, it is not surprising that
with a lower tem perature, the extent o f glycation is comparatively lesser than at a higher
temperature.
The presence o f antimicrobial agents also affects the extent o f glycation. The
correlation betw een the extent o f glycation o f lysozym e-glucose m ixture that contained
sodium azide and the duration o f incubation showed an overall linear increase (Figure
5.5b). The presence o f 0.01% sodium azide served to destroy the micro-organisms or
bacteria that were present in the system. The linearity o f the correlation betw een the
extent o f glycation suggested that glycation occurred at an increasing pace but very slow.
The lower extent o f glycation as com pared to that o f the samples without sodium azide
also suggested that minimal conformational change occurred with the presence o f sodium
azide, allowing only the lysyl groups that were exposed in the native state o f lysozyme to
react with glucose.
The extent o f glycation o f the high-pressure treated samples is shown in Figure
5.6. The optimum glycation was reached after 8 hours o f pressurization. This optimum
level accounted for 60% lysyl groups being glycated, which is about 3 times as much as
subjecting the lysozyme-glucose m ixture to 6 days o f incubation. It has been reported that
56
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
high pressure accelerates the formation o f the initial condensation product at the early
stage o f M aillard reaction - the Amadori stage (Tamaoka et al., 1991; Issacs and Coulson,
1996; B ristow and Issacs, 1999). A fter 8 hours, the extent o f glycation o f lysozyme
decreased. This may be due to the reversibility o f the Schiff base or Amadori
rearrangem ent, releasing the primary amino groups to be detected by the fluorescamine
assay (Y aylayan and Huyghues-Despointes, 1996; Davidek et al., in press). Another
possible m echanism could be the presence o f minute amounts o f oxygen in the sealed
plastic bags containing the samples that could allow for the browning reactions (Issacs
and Coulson, 1996) and the form ation o f deoxyglucosones upon degradation o f the
Am adori com pound with the release o f the free amino groups (Zhao, 2001) to be able to
react with fluorescamine, therefore, resulting in lower extent o f glycation. A correlation
betw een the extent o f glycation and the duration o f pressurization was not possible since
the optimum point o f glycation occurred at the first m easured data point. Future work
w ith more data points before 8 hours should be collected, so as to generate a correlation
betw een the extent o f glycation with the duration o f pressurization.
(a)
W ITHOUT SOD IUM A ZID E
(b)
60
W IT H SO D IU M A ZID E
50
= -0.2017X2 + 4.4574x + 0.1083
40
0.9925
u 30
c
x
y=0.4Q69x + 0.4092
20
-
10
-
R2 = 0.9231
to
0
2
4
6
8
10
0
12
Time under regular incubation (days)
2
4
6
8
10
12
Time under regular incubation (days)
Figure 5.5
Correlation o f the extent o f glycation o f lysozym e-glucose m ixture that
have been subjected to regular incubation at 30°C over a period o f 12 days when (a)
0.01% sodium azide was absent; and (b) 0.01% sodium azide was present. Both are
compared to the initial extent o f glycation o f the day 0 sample.
57
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
(b)
60 -
(a)
50 ^
= -0.2Q17X2 + 4.4574x + 0.1083
R2 = 0.9925
40 ^
30 -
20 j
1 0 -i
0
0
40
50
20
30
10
Time u n d er high p re ssu re (hours)
2
4
6
8
10
12
Time under regular incubation (days)
Figure 5.6
Extent o f glycation o f lysozyme-glucose m ixture that have been subjected
to (a) 400 M Pa o f HHP at 30°C over a period o f 48 hours; and (b) regular incubation at
30°C over a period o f 12 days, both are com pared to the initial extent o f glycation o f the
day 0 sample.
The extent o f glycation o f microwave-treated samples, over a period o f 60
minutes at 50°C, varied between 30 to 43% w ith the maximum glycation reached after 30
m in as shown in Figure 5.7a. This variation suggests that reversible reactions took place
upon reaching a certain m aximum extent o f glycation. The glycated lysozym e seemed to
undergo reversible reactions as observed in the samples that were being subjected to HHP,
allowing for more free amino groups that got released to react with fluorescamine. It
m ight possible that conformational changes due to folding and unfolding o f the proteins
took place throughout the 60-minute microwave irradiation period. The large standard
deviation o f the triplicates obtained for the 30-minute sample (±10.5% ) suggested that the
protein underwent random conform ational changes, affecting the availability o f lysine to
react with glucose, and hence affecting the overall extent o f glycation generated. Overall,
glycation reached a m aximum o f around 40% with 20 minutes o f m icrowave irradiation,
which is comparable to two times the extent o f glycation as when the lysozym e-glucose
mixture was subjected to regular incubation at 30°C under standard conditions. To avoid
58
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
building up o f excessive pressure during the microwave process, the centrifuge tubes
containing the samples were not capped. A s a result the samples m ight have experienced
some m oisture loss to the surrounding as the reaction progressed, w hich in turn decreases
the availability o f water as polarizable molecules for microwave absorption, as suggested
by Yeo and Shibamato (1991). Therefore, there is a possibility that the free lysyl groups
did not have sufficient energy to bind with the free glucose again upon the reversible
reactions o f ARP, resulting in a decrease in the extent o f glycation as the treatment time
increased.
The extent o f glycation o f lysozyme samples being incubated from 10 to 60
m inutes at 50°C was also studied to eliminate the effect o f tem perature from microwave
irradiation on glycation. Incubating the samples from 10 to 50 m inutes did not show any
sign o f protein denaturation and glycation, while w hen the incubation time at 50°C
increased to 60 minutes, there was a minimal 8% o f glycation. In other words, the extent
o f glycation found on the microwave-treated samples can safely be assum ed that it was
the effect o f m icrowave irradiation and not the temperature.
From the data obtained, there seems to be a non-linear (polynom ial) correlation
between the extent o f glycation and the time o f m icrowave irradiation (Figure 5.8). The
polynomial correlation suggested that optim um glycation occurred at around 23 minutes
under the experimental conditions, and the extent o f glycation after this plateau would
decrease upon prolonged exposure to microwave irradiation, suggesting the formation o f
irreversible crosslinks. Future work on validating the correlation betw een the extent of
glycation and the exposure time o f Am adori product to focused m icrowave irradiation
59
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
should be perform ed, In addition, the duration o f microwave exposure should decrease to
m inutes to estim ate the changes before 10 minutes, as to obtain a clearer picture in
generating glycation w ith microwave irradiation.
80
(a)
60 i
50
0.2017x2 + 4.4574x+0.1083
R2 = 0.9925
£ 40
O
30
tsui
*.
© 20
1
2
10
0
0
10
20
30
40
50
60
0
Microwave time (minutes)
2
4
6
8
10
12
Time under regular incubation (days)
Figure 5.7
Extent o f glycation o f lysozyme-glucose m ixture (a) subjected to focused
m icrowave irradiation at 50°C over a period o f 60 minutes; and (b) subjected to regular
incubation at 30°C over a duration o f 12 days, both are com pared to the initial extent o f
glycation o f the day 0 sample.
60
50
c
40
t
30
OJ
^
20
y = Q.QOIx - G.1236x + 4 .1 4 5 6 x + 0 .3 8 9
c
©
R2 = 0 .9 9 1 2
0
10
20
30
40
50
Microwave time (minutes)
Figure 5.8
Correlation o f the extent o f glycation o f lysozym e-glucose mixture versus
the duration o f m icrowave irradiation at 50°C.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
5.1.3
C O M P A R IS O N OF T H E EFFECT O F T H E T W O TREATMENTS ON
G L Y C A T IO N O F LY SO ZY M E
In com paring the different types o f treatments, high pressure seems to cause the
least protein destruction even at prolonged exposure time like 48 hours at 400 M Pa, and
at the same tim e achieving higher extent o f glycation. For example, three times more
glycation was observed in subjecting the samples to high pressure for 8 hours as
com pared to incubating the samples at 30°C for 6 days; or 2 times more glycation in 8
hours as com pared to 10-minute o f m icrowave irradiation. M icrowave irradiation could
generate around 30 to 40% glycation w ithin 60 m inutes. However, the protein loss was
slightly higher for the m icrowave-treated samples. There was a non-linear (polynomial)
correlation betw een the microwave time and the extent o f glycation that can be achieved,
w ith an optim um glycation o f 43% at around 23 m inutes, which was around 2 times as
m uch as incubating the samples for 6 days at 30°C.
On the other hand, regular incubation at 30°C, causes the highest extent o f protein
destruction (around 50%) and only 20 to 25% o f glycation could be achieved. However,
addition o f 0.01% sodium azide to the samples prevented damage or denaturation to the
protein, but it did not seem to promote glycation.
5.2
E F F E C T O F HHP AND MICROWAVE IRRADIATION ON USA
BSA (66,000 Da) being a larger protein than lysozyme (14,300 Da) is more prone
to degradation during thermal processing. It has been reported that when proteins and
reducing sugars are heated, the corresponding solubility o f the proteins would be reduced
61
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
along with occurrence o f gelation. (Hill et a l, 1992; Yowell and Flurkey, 1986; Kaye et
a l, 2001). G elling occurred with BSA samples that had been subjected to HH P and
focused m icrow ave irradiation, in which, gelation was defined by Ferry (1948) is a twostep process with unfolding or dissociation o f the protein m olecules followed by
association or aggregation reactions.
To carry out the tests to determine the protein content and the glycated lysyl
groups, 12% SDS solutions were added to the gels and the subsequent soluble protein
content was estimated from the Lowry test to calculate the extent o f glycation.
5.2.1
L O W R Y T E ST
Samples o f BSA that were incubated in the oven at 30°C are the only samples that
did not gel, and remained in solution. The Low ry test, did not detect m uch protein loss
for samples incubated with glucose; while the corresponding controls had approximately
10% loss in the protein content (Figure 5.9). The presence o f glucose therefore seems to
act as a protectant. The overall protein content o f the BSA controls was lower than that o f
the BSA-glucose mixtures, and this further suggested the role o f glucose in the mixture as
a stabilizer to the protein. This observation also agrees well with a previous study in the
reaction between human serum album in and glucose (Brimer et a l, 1995). According to
this study, the mild conditions o f incubation or storage o f food proteins in the presence o f
reducing sugars m ainly affect lysine, thereby protecting the integrity o f the other amino
acids from crosslinking through lysyl residues (H arrell and Carpenter, 1977). Therefore,
due to M aillard reaction, the form ation o f isopeptides is retarded (Otterbum , 1989).
62
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Isopeptide bond form ation is one o f the comm on types o f crosslinks that can happen in
proteins, especially in proteins that are rich in lysine since it can form a bond between Its
y-amino group and an adjacent carboxylic acid (Lundblad, 1995). Furthermore, the
standard deviations o f the triplicates o f the protein controls throughout the 12-day
incubation w ere high, as compared to the glucose-containing samples. This further
suggested that glucose acts as a stabilizer to the protein in preventing random
conform ational changes throughout the incubation period. The above observation seems
in contradiction with a previous study in which, the percentage o f soluble BSA o f the
BSA-glucose m ixture incubated in the presence o f oxygen experienced a gradual loss
w ith time (Yeboah et al., 1999). A possible explanation in the discrepancy between the
two experiments is the presence o f oxygen. Oxygen is an im portant factor in governing
the protein loss since it allows for oxidation reactions. Samples in this study were placed
in m icro-centrifuge tubes with caps closed upon incubation, therefore securing minimal
amounts o f oxygen for degradation. In addition, differences on the physical state o f the
proteins upon incubation (dry versus solution) m ay also affect the rate o f M aillard
reaction since m oisture content or water activity o f the system is a critical factor that has
an effect on many o f the pathways o f M aillard reaction and the extent o f crosslinking
(Wu etal., 1990; Ames, 1998).
63
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
100
c
80
?
60
Z
40
8
20
CL
3
- ♦ - w it h glucose
—•— protein controls
0
2
4
6
8
10
12
Time under regular incubation (days)
Figure 5.9
Percentage o f water soluble BSA rem ained in the samples upon being
subjected to regular incubation at 30°C over a period o f 12 days, as compared to the
initial am ount o f BSA at day 0.
Figure 5.10 shows the percentage o f soluble BSA in the BSA-glucose mixtures
and the BSA controls that have been subjected to HHP over a period o f 48 hours.
Significant losses in the protein content o f around 90% and 70% for the controls and the
glucose-containing samples respectively were observed over the 48 hours o f incubation.
Previous studies conducted on the denaturation o f BSA under high pressure have found
no signs o f denaturation even at 400 M Pa (Hayakawa et al., 1992; Aoki et a l, 1966);
similarly, when ovalbum in was subjected to pressures up to 800 M Pa in the presence o f
sucrose also showed no signs o f denaturation (lam etti et al., 1998). W hile in the studies
conducted by Galazka et al. (1997a,b) aggregation o f BSA was observed upon
pressurization. A possible explanation for the discrepancies betw een different studies
could be attributed to the differences in the type o f saccharides and in the concentration
o f the BSA solutions used since more concentrated solutions are m ore prone to decreased
solubility upon HHP processing, leading to gelation. In addition, time o f exposures to
64
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pressure is also a factor in governing gelation, with a longer exposure time allowing the
protein or protein m ixture to gel and crosslink. In comparing the glucose-containing
samples w ith the protein controls, the former seemed to be m ore capable o f retaining the
integrity o f the protein due to the occurrence o f M aillard reactions, resulting in higher
amount o f soluble protein after being processed. Nonetheless, as the processing time
increased, m ore conformational changes occurred, .leading to the occurrence o f
denaturation or irreversible crosslinks. It seems that the protein with the exposed side
chains reacted with each other, inducing reversible crosslinks as in the samples that had
been subjected to regular incubation. In addition, the standard deviation o f the 8-hour
pressurized sample that contained glucose was high, suggesting that the protein
underw ent significant conformational changes and random cross-linking. Protein controls,
on the other hand, did not show random ized crosslinking with sm aller standard deviations.
Nonetheless, results showed that the protein content o f both the glucose-containing BSA
and the BSA alone reduced significantly upon subjection to 400 M Pa o f high pressure,
despite the possible types o f reversible crosslinks that could be formed. In addition, all
the BSA samples gelled upon being subjected to high pressure. Gels were solubilized in
SDS solutions for Lowry test and fluorescamine assay. The fact that SDS did not
solubilize the gels effectively and efficiently upon vortexing and centrifuging, could also
lead to underestim ation o f the protein content. Future work has to be done on more
detailed amino acid analyses to detect the status o f the amino acids in the sample. In
addition, shorter HHP processed times such as m inutes should be studied so as not to
destroy the protein totally while trying to promote M aillard reaction.
65
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
100
with glucose
protein controls
c
ok .
a
5
JS
a
o
»
0
10
20
30
40
50
Time under high pressure (hours)
Figure 5.10
Percentage o f soluble protein o f BSA samples subjected to HHP at 30°C
as compared to the initial amount o f the protein at day 0.
Figure 5.11 showed the percentage o f soluble protein content o f the BSA samples
that have been subjected to focused m icrowave irradiation over a period o f 60 minutes.
W ith the m icrowave treatment, the damage to the proteins was less, compared to those
treated with high pressure. M icrowave heating o f proteins has been reported to have
reduction in their water solubility (Yowell and Flurkey, 1986; Dowdie and Biede, 1983).
Once again, the protein content o f the glucose-containing samples was higher than that o f
the controls, further confirming the role o f glucose as a stabilizer in protecting the
proteins from further crosslinking with other amino acids o f the protein during
microwaving. However, the protein content o f the BSA-glucose mixture kept decreasing,
suggesting that a major amount o f the protein got denatured irreversibly with loss in
solubility upon prolonged exposure to m icrowave irradiation. At the same tim e, the
protein content o f the controls encountered almost total loss o f the protein’s solubility
throughout the process o f m icrowave irradiation, suggesting the occurrence o f
66
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irreversible crosslinks. Furthermore, the large standard deviations o f the samples that
contained glucose suggests that the conformational changes experienced by the protein
were not as uniform as the changes that were experienced by the BSA in the absence o f
glucose.
Overall, BSA seems to be a protein not suitable for high energy exposures. High
pressure treatm ent caused the greatest damage to BSA, followed by microwave
irradiation, because as the BSA samples are being subjected to high energy exposures,
competing reactions such as crosslinking o f the proteins can take place more readily.
Note that isopeptide bond form ation is one o f the common type o f crosslinks that could
occur in proteins that have m any lysyl groups, and BSA is one o f the proteins that
contains numerous lysine groups (66 lysines), as well as glutamic and aspartic acids,
which provides the carboxylic group for the lysine to crosslinking (Brown, 1975;
Patterson and Geller, 1977; M cGillivray et a l, 1979; Reed et al., 1980; Hirayam a et al.,
1990).
While for the samples that were being incubated at 30°C, the integrity o f the
protein was maintained, particularly for the samples that contained glucose upon
incubation, confirming the role that glucose acts as a protectant in inhibiting crosslinking,
while promoting M aillard reaction.
67
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
100 K
80 -
c
with glucose
protein controls
1
®
a
40 -
1
55
20 -
60 *
K
0
10
40
20
30
Microwave time (minutes)
50
60
Figure 5.11
Percentage o f soluble BSA in samples subjected to focused microwave
irradiation from 10 minutes to 60 m inutes at 50°C, as compared to the initial amount o f
the protein at day 0.
5.2.2
F L U O R E S C A M IN E ASSAY
Figure 5.12 shows the correlation between the extent o f glycation in the
incubation samples o f BSA for 6, 8, 10 and 12 days. Com pared to the unm odified BSAglucose sample at day 0, around 33% o f the BSA got glycated on day 6, and the extent o f
glycation minimally increased up to 10 days o f incubation, in agreem ent w ith a previous
study on the reactivities o f BSA (Yeboah et al., 1999). Upon prolonged incubation, it
seemed that the ARP underw ent reversible changes in releasing the sugar moieties from
the lysine residues. In addition, the non-linear (polynom ial) correlation suggested that
glycation reached a m axim um at around 9 days with about 35% glycation
which
represents around 22 lysyl groups (out o f a total o f 61) (see Table 4.17 for calculated
results and Appendix A3.2 to A3.4 for steps in calculating the extent o f glycation).
68
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60
g 30
a
= -0.4268X2 + 7 .8 1 34x * 0.1662
R2 = 0.9949
*5 2 0
4
2
0
8
6
10
12
T im e under re g u la r in c u b a tio n (d ay s)
Figure 5.12
Correlation o f the extent o f glycation o f BSA-glucose m ixture versus time
o f incubation at 30°C relative to day 0 BSA-glucose sample.
60
y = -0.4268X2 + 7.8134x + 0.1662
R2 = 0.9949
50
y = -0.0291 x2 + 1.6843x + 1.2339
R2 = 0.9724
40
30
o>
as
20
10
0
10
20
30
40
0
50
0
Time under high pressure (hours)
4
6
8
10
12
2
Time under regular incubation (days)
Figure 5.13
Correlation o f the extent o f glycation o f BSA-glucose m ixture versus the
tim e o f (a) HHP treatm ent at 30°C over a period o f 48 hours; and (b) regular incubation at
30°C over a duration o f 12 days, both are com pared to the initial extent o f glycation o f
the day 0 BSA-glucose sample.
W hen subjecting the BSA-glucose m ixture to HHP over 48 hours, the correlation
o f the extent o f glycation versus the time o f the samples under pressure obtained is shown
in Figure 5.13a. This figure indicates that with 8 hours o f pressurization, BSA only
attained some 12 to 15% o f glycation, and the extent o f glycation increased to around
69
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
24% for the 24-hour treatment, and reverted back to 15% for the 48-hour treatment. A
possible explanation for the lower extent o f glycation as com pared to the incubation
samples is that the glucose underwent rapid m utarotation from pyranose to furanose with
the high pressure, leaving very small amounts o f the free aldehydes which serves as
intermediate for the conversion in the solution (Andersen and Gronlund, 1979; Andersen
et al., 1984), in which, the free aldehyde o f glucose is the form that the lysine reacts with
upon glycation. It seems that the rapid m utarotation slowed down the reaction between
glucose and the lysine groups, and crosslinking o f the proteins occurred at a faster rate
than the lysine groups reacting with the aldehyde groups o f the glucose.
Figure 5.14a shows the extent o f glycation o f the BSA-glucose m ixture upon
microwave irradiation at 50°C over a period o f 60 minutes. The extent o f glycation
decreased after reaching a m aximum at 10 minutes o f irradiation. At this time 30% o f the
soluble
BSA
remained,
suggesting
that
70%
o f the
protein
were
denatured.
Fluorescamine assay estimated 17% glycation, representing an average number o f 10
glycated lysyl groups. W hile on the other hand, regular incubation had around 100% o f
soluble protein upon 6 days o f incubation, while attaining around 30% o f glycation. This
suggested that m icrowave irradiation provided an accelerating effect on both the M aillard
reaction and protein denaturation as com pared to the samples being subjected to regular
incubation, and this is in agreement with the observation o f Villamiel et al. (1996).
70
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(b)
60 i
(a)
y = -0.4268x2 + 7.8134x + 0.1662
3- 50 H
50 J
R2 = 0.9949
40 -i
t
30os
10 0
UJ
10
20
30
40
50
60
Microwave time (minutes)
0
4
6
8
10
12
2
Tin® under regular incubation (days)
Figure 5.14
Extent o f glycation o f BSA-glucose m ixtures (a) subjected to microwave
irradiation at 50°C over a period o f 60 minutes; and (b) subjected to regular incubation at
30°C over a period o f 12 days, both are com pared to the initial extent o f glycation o f the
day 0 BSA-glucose sample.
The extent o f glycation observed under m icrowave irradiation decreased as the
m icrowave time increased, and followed the same trend as the high pressure treated
samples betw een 24 and 48 hours o f treatment. This observation suggests that the protein
denaturation happens concurrently as the M aillard reaction. However, since the
processing time with microwave irradiation is comparatively shorter than that of the high
pressure treatment, denaturation was not as extensive as the HHP treated samples and
more o f the lysine groups in the proteins were free from being crosslinked and could
react with the glucose moieties. The standard deviation o f the 60-minute sample is
comparatively higher, suggesting the occurrence o f random conform ational changes o f
the protein. The non-linear (polynomial) correlation between the extent o f glycation and
the microwave time shown in Figure 5.15 suggested that optimum glycation can be
obtained with short durations o f m icrowave exposure o f around 20 minutes, and
prolonged exposure would only result in denaturation o f the protein.
71
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60
-i
50
y = -0.01 Sx2 + 0.809x + 0.7674
40
I0»
30
'S
20
0.7992
€
10
10
20
30
40
M icrow ave tim e (m in u tes)
50
60
Figure 5.15
Correlation o f the extent o f glycation o f BSA-glucose m ixture versus the
tim e under m icrowave irradiation.
5.2.3
C O M P A R IS O N O F T H E E F F E C T OF THE T W O T R E A T M E N T S ON
G L Y C A T IO N O F BSA
Comparison o f the effect o f different types o f treatm ents on BSA indicates that
BSA is very susceptible to crosslinking or other mechanisms that m ay damage the
proteins. The results also indicate that high energy exposure regardless of duration
severely damages the protein. An 8-hour HHP treatm ent at 400 M Pa could only attain
one fourth the extent o f glycation to that o f 6-day regular incubation at 30°C; at the same
time, a 10-minute microwave irradiation could only attain half o f the extent o f glycation
to that o f a 6-day regular incubation, furthermore, a 10-minute o f m icrowave irradiation
attained comparable extent o f glycation to that o f 8-hour HHP treatment. Future work
should be done on obtaining optim um extent o f glycation with the least damage to the
proteins with shorter durations o f high energy exposures.
72
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5.3
OVERALL
COMPARISON
OF
THE
EFFECT
OF
DIFFERENT
T R E A T M E N T S ON BSA AND LYSOZYME
In com paring the effect o f the two high energy exposures on the two proteins, it
can be concluded that lysozyme is the protein that will not undergo m uch degradation
upon prolonged high energy treatments with appreciable extent o f glycation from 30 to
60%. W ith around 80% o f soluble protein remaining after HHP treatm ent, extent o f
glycation o f the soluble protein could reach up to 60%. For the m icrowave-treated
samples, 60% o f the lysozyme rem ained active upon processing attaining 40% glycation.
Incubating the lysozyme-glucose solution at 30°C over a period o f 12 days encountered
the most dam age to the protein, leaving only about 40% o f the protein active, and
generated about 20% o f glycation. On the other hand, BSA was very susceptible to high
energy exposure and experienced high protein loss. BSA-glucose solution experienced
around 70 to 80% protein loss upon treatment with HHP and around 80 to 90% with
microwave irradiation. The protein content o f the m icrowave-irradiated samples was
shown to decrease as the m icrowave time increased. However, the protein content o f the
BSA-glucose samples experienced negligible loss upon incubation for up to 12 days at
30°C. The extent o f glycation was also negligible for the samples treated with high
energy exposures, with only 10 to 20% o f the soluble protein achieving glycation. On the
other hand, while glucose did not protect the integrity o f lysozyme, however it did so to
BSA. Less BSA got denatured upon high energy exposure compared to the controls with
no glucose added. The addition o f sodium azide served to protect lysozyme from
microbial degradation during incubation, nonetheless, it did not seem to promote
glycation.
73
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From a practical point o f view, high energy exposure is not suitable for generating
glycated B SA . Even w ith a 100% soluble protein rem aining under regular incubation,
only 35% w as glycated. This suggests that a large protein with m any lysyl groups is hard
to glycate, because the lysyl groups remain hidden w ithin the protein m atrix and are not
exposed to the environment for glycation. O n the other hand, lysozyme, being a
com paratively smaller protein with only 14,300 Da and 6 lysyl groups, it can more
readily undergo conformational changes to expose the lysyl groups for glycation.
Subjecting the lysozyme-glucose mixture to high energy exposures can actually
accelerate the reaction betw een glucose and the lysyl groups, increasing the extent o f
glycation.
5.4
FUTURE STU D IES
To generate a better picture o f glycation o f proteins, HHP processing and
microwave irradiation experiments could be perform ed at shorter reaction times so as to
obtain information on the initial progress o f glycation. In addition, operating parameters
such as the m agnitude o f HHP applied, tem perature o f the m icrowave and HHP reactions,
should be investigated so as to better promote M aillard reaction effectively in a relatively
short period o f time. M ore detailed analyses such as amino acid analysis could be done
to reveal the actual amino acid content o f the proteins upon being treated, with the extent
o f glycation to be estimated more precisely. Spectroscopic studies o f the treated proteins
should be carried out to validate the changes in the conform ation o f the proteins upon the
different types o f treatments.
74
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6
A N A LY SIS O F G L Y C A T E D P R O T E IN S BY PY R O L Y SIS-G C /M S
6.1
IN T R O D U C T IO N
There are numerous methods available for detecting the presence o f glycated
proteins. M ethods such as TBA method, periodate oxidation, borohydride reduction,
N B T colorim etric assay, ferricyanide test, Elson-M organ test, etc. are some o f the known
m ethods for detection o f Amadori product or glycated proteins. Separation-based assays
using chrom atography can be used to separate glycated proteins from unglycated species
based on the charges o f the species involved. Glycated proteins can also undergo
hydrolysis and form furosine which can be separated by HPLC and then detected by
measuring the corresponding absorbance at 280 nm (Furth, 1988). Fluorescence
generated from the reaction o f lysine with fluorescamine can as well be m easured at 390
and 475 nm (em ission and excitation wavelengths) to assay the extent o f glycation
(Yaylayan et al., 1992). Other methods such as using infrared spectroscopy to detect and
quantitate the open chain or keto forms o f Am adori products, and subsequently used to
infer the amount o f Amadori product present, is a com paratively fast technique, however,
lack o f sensitivity m ay hinder its use as a practical m ethod (Yaylayan and HuyghuesDespointes, 1994). Other spectroscopic methods such as GC/MS (gas chromatography mass spectroscopy) are available for detection o f glycated proteins, that require the
samples to be derivatized or hydrolyzed prior to analysis, so that the compounds can be
injected into the colum n to be analyzed. Com m on mass spectroscopic methods used
nowadays such as ESI-M S (Electrospray ionization m ass spectroscopy) and M ALDI-M S
(matrix assisted laser induced ionization m ass spectroscopy) require less sample
pretreatm ent steps as compared to regular GC/M S (Yeboah and Yaylayan, 2001). With
75
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the use o f m ass spectroscopic methods, the intact glycated proteins can be analyzed
qualitatively and quantitatively. Changes in the conformation o f the proteins, the
glycation sites and the distribution profile o f glycoforms upon glycation o f the proteins
are some o f the qualitative aspects that can be m onitored by ESI-M S (Yeboah et a!.,
2000).
All the above methods can be used to detect glycation in proteins, nonetheless,
drawbacks for the chemical methods include susceptibility to interference by smallm olecule contaminants or protein-bound groups in unpurified preparations, as well as
enzymatically bound sugars, affecting the true readings o f the test (Furth, 1988). The time
consuming sample preparation steps for the spectroscopic methods such as derivatization
or hydrolysis prior to analysis make them inconvenient to be used. Pyrolysis-GC/M S is
therefore an alternative m ethod that can be considered to detect protein glycation, since it
is ideally suited to handle non-volatile compounds without tedious sample preparation.
This proposition is based on the fact that any hexose moiety, whether free or existing as
ARP such as in glycated proteins, can be decom posed to produce the characteristic 2,3dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one peak (M+ at m/z 144) upon pyrolysis
(Figure 6.1) at temperatures higher than 100°C (Yaylayan and Keyhani, 1996).
76
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Pyrolysis
N
H
250°C
HO
OH
HO
OH
O
OH
m/z 144
G lycated protein
Diagnostic ion for the presence o f
Amadori compound on proteins
Figure 6.1
The degradation o f Amadori rearrangement product upon pyrolysis with
the production o f the ion at m /z 144.
The breakdow n o f a complex structure into smaller units for identification has
been regularly used as an analytical technique (Mackillop, 1968). Pyrolysis is a technique
that involves the breakdow n o f large complex m olecules into sm aller and more
analytically useful fragments by applying direct heat. Pyrolysis is able to generate
fragments in a reproducible m anner when the heat energy that is applied to the molecule
is greater than the energy o f its specific bonds. Therefore, the bonds will dissociate in a
predictable and reproducible way for analysis (W ampler and Levy, 1987). Coupling the
pyrolyzer with analytical tools such as GC/M S or FTIR spectroscopy serves to interprete
the identity o f these fragments and to understand the structure o f the original
macromolecule.
Com pared
to
conventional, methods
of
lengthy
extractions
or
derivatization in analyzing insoluble m aterials such as wood, rubber, plastic, etc. by GC,
MS and FTIR, pyrolysis is a much faster technique that requires small am ount o f samples
with
minimum
sample
preparation
for
reproducible
results
qualitatively
and
quantitatively (Goodacre and Kell, 1996; Yaylayan and Keyhani, 1996). Pyrolysis-
77
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GC/M S m ethodology therefore, would serve as a rapid and accurate technique of
detecting the am ount o f intact ARP present using the MT ion at m /z 144 as a diagnostic
marker. The objective o f this part o f the study is to investigate the use o f the M + ion at
m /z 144 as a universal m arker for glycated proteins by Pyrolysis-GC/M S assay, and to
generate a subsequent correlation in glycation with the duration o f incubation.
6.2
M A T E R IA L S AND M E T H O D S
6.2.1
MATERIALS
Glycated hum an albumin (GHSA, 95 % protein, 2.7 mol hexose/mol), human
albumin (HSA), lysozyme, D-glucose and silica gel obtained from Sigma Chemicals (St.
Louis, M O., USA), glycated lysozymes (LG-2, LG-5, L G -10 and L G -14) were generated
according to the m ethod o f Yeboah et al. (2000). Lysozyme samples treated with 8 hours
o f HHP and 10 m inutes o f microwave irradiation (see Section 3.5 and 3.6.1) were freezedried for further use in this study. The quartz tubes w ith 0.3 mm thickness used were
obtained from CDS Analytical Inc. (Oxford, OA). A Varian Saturn 2000 GC/MS
interfaced to a CDS Pyroprobe 2000 unit (CDS Analytical Inc., Oxford, PA) with a fused
silica DB-5 MS colum n (50 m length x 0.2 mm i.d. x 0.33 pm film thickness; J&W
Scientific) was used for the Pyrolysis-GC/M S assay.
6.2.2
SA M PL E PREPARATION
6.2.2.1 IN T E R F E R E N C E OF F R E E G L U C O S E WITH PY R O L Y S IS ASSAY
Glycated protein mixtures prepared from 8 hours o f HH P and 10 m inutes o f
focused microwave irradiation were freeze-dried for pyrolysis. Table 6.1 shows the
78
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amount o f protein, glycated protein, glucose and silica gel added to individual batches
w ith thorough grinding. The two proteins used (lysozyme and HSA) were m ixed with
silica gel w ith or without glucose. One mg o f solid sample from each batch was placed in
a quartz tube for pyrolysis which contained 0.8 mg o f the protein involved. In addition,
glucose (1 m g) was pyrolyzed separately. Samples were introduced into quartz tubes
plugged w ith quartz wool and pyrolyzed.
6.2.22 C O R R E L A T IO N
O F THE IN T E N S IT Y O F THE PY R O L Y SIS­
G E N E R A T E D M A R K E R PEAK FROM C O M M E R C IA L L Y A V A ILA BLE
G L Y C A T E D HUMAN SE R U M A L B U M IN (G H SA ) W IT H T H E A M O U N T
O F PROTEIN
Seven m ixtures o f commercial GHSA and silica gel were prepared with thorough
grinding, w ith increasing concentrations o f GHSA in each batch (i.e. 1 or 2 mg o f the
solid m ixture from each batch contained GHSA ranging from 0.1918 mg to 1.4074 mg,
with the rem aining amount made up by silica gel). Two replicate pyrolysis were
performed from each batch, and the 7 concentrations o f the m ixtures were tabulated in
Table 6.2. Samples introduced into quartz tubes were plugged with quartz wool and
pyrolyzed.
6 2 .2 3 C O R R E L A T IO N O F T H E IN T E N S IT Y OF PY R O L Y S IS -G E N E R A T E D
M A R K E R P E A K FROM P R E P A R E D G L Y C A T E D LYSOZYME WITH
THE D E G R E E OF G L Y C A T IO N
LG-2, LG-5, L G -10 and L G -14 were glycated lysozyme generated by incubating
lysozyme at 50°C with glucose for 2, 5, 10 and 14 days respectively according to the
method o f Yeboah et al. (2000), followed by dialysis to rem ove unreacted sugars and
freeze drying. Each o f the samples above (3.2 mg) was m ixed thoroughly with silica gel
79
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(0.8 mg) and the mixture (1 mg) was introduced into quartz tube plugged with quartz
wool for pyrolysis.
Table 6.1
The constituent o f the samples duly prepared for pyrolysis. Lysozyme
sample treated with 8 hours o f HHP is designated by HHP, and sample treated with 10
m inutes o f m icrowave irradiation is designated by MW.__________ ____________________
W eight of glucose
W eig h t of silica gel
W eight o f protein
B atch n a m e
(m g)
(mg)
_
3.2
0.8
HSA
3.2
0.3
0.5
HSA + glucose
.
0.4
1.6
GHSA
3.2
0.8
Lysozyme
3.2
0.3
0.5
Lysozyme + glucose
.
1.0
1.0
Glucose
0.8
0.2
HHP
0.2
MW
0.8
Table 6.2
The weight o f GHSA and silica gel in the m ixture prepared for pyrolysis.
W eight o f G H SA
W eig h t o f silica gel A m o u n t of m ix tu re
B atch #
in mixture
in m ix tu re
pyrolyzed
(mg)
(g)
(g)
0.0014
1
0.0059
1.0
0.0012
0.0021
2
1.0
3
0.0019
0.0028
1.0
0.0026
0.0016
4
1.0
0.0035
5
0.0009
1.0
0.0032
0.0021
6
2.0
0.0038
7
0.0016
2.0
6.2.3
M ETHODS
In all pyrolysis experiments, the pyroprobe interface tem perature was set at 250°C
at a heating rate o f 50°C m s'1 with a total heating time o f 20 sec. The mass range
analyzed was 29 -
300 amu. The volatiles generated were sent to the sample
preconcentration trap (SPT) at -30°C and trapped, followed by desorption at 250°C . The
80
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voiatiles were injected with a split ratio o f 260:1, and were analyzed under delayed
pulsed pressure. Pressure was increased from 1 psi to 70 psi at a rate o f 400 psi min"3 and
held for 2 min, then dropped to 2 psi at a rate o f 400 psi m in'3 to establish a constant flow
o f 1.5 mL m in'1. The initial tem perature o f the colum n was set at -5°C for 12 minutes,
and then increased to 50°C at a rate o f 50°C m in'3; the tem perature was then further
increased to 250°C at a rate o f 8°C m in '1 and kept at 250°C for 5 min. The ions formed
from the ARP were identified and quantified w ith the relative intensity through library
search o f NIST and SATURN libraries o f the SATURN software. Autom ated Mass
spectra Deconvolution and Identification System (AMDIS), Version 2.0 - DTRA/NIST
1999) is the program that was used for deconvolution o f components detected from
Pyrolysis-G C/M S. Curve fitting equations were generated by Microsoft® Excel (2002)
software package.
6.3
R E SU L T S AND D ISC U SSIO N
6.3.1
IN T E R F E R E N C E O F F R E E G L U C O S E W IT H P Y R O L Y SIS ASSAY
Pyrogram o f pure glucose serves as a guide in confirming the retention time o f the
peak generated from 2,3-dihydro-3,5-dihydroxy-6-m ethyl-4H-pyran-4-one (M + at m/z
144), which is present at around 26.20 min under the experimental conditions used (Peak
denoted by I in Figure 6.2). In addition, the m ajor peak detected in the pyrogram o f pure
glucose was hydroxym ethylfurfural (HMF; peak denoted by H in Figure 6.2), which can
be formed during dehydration or degradation o f free hexoses (Kroh, 1994; Anam and
Dart, 1995). Detection o f HMF in the pyrolysis m ixtures can therefore be used to indicate
81
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the presence o f free glucose, since Amadori products do not generate significant intensity
o f HMF (Yaylayan and Keyhani, 1996).
M C o u n ts
J i .wi s.
..
M C o u n ts
m inutes
Figure 6.2
Pyrograms o f glucose showing (a) total ion current and (b)
Extracted Ion chrom atograms (m/z 144 and m /z 126). Peaks containing the
M + ion at m/z 144 and the ion from HMF at m/z 126 are designated by I
and H respectively.
82
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kCounts ~
300 1
200 i
100 -
j'.J: !
^j. U.i
kCounts -
300 200 4
100
-
kCounts ;
300 ~
200
-
100
-
J J
kCounts
300
200
-
100
-
JtJuX,
«LI
kCounts ;
300 200 -J
100 -
10
20
30
40
minutes
Figure 6.3
Pyrogram s showing the total ion current o f (a) HSA; (b) GHSA; (c)
HSA with glucose; (d) iysozyme; (e) lysozyme with glucose. Peaks containing the
M + ion at m/z 144 are designated by I in the pyrograms.
83
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kCounts
12.5
10.0
7.5
5.0
2.5
0.0
kCounts
10.0
7.5
5.0
2.5
QJ3
kcounts
10.0
7.5
5.0
2.5
0.0_
kCounts
12.5
10.0
7.5
5.0
2.5
0.0
kCounts
10.0
7.5
5.0
2.5
0.0
m inutes
Figure 6.4
Extracted Ion chromatograms (m/z 144 and m/z 126) o f (a) HSA; (b)
GHSA; (c) HSA with glucose; (d) lysozym e; (e) lysozym e with glucose. Peaks
containing the M + ion at m/z 144 and the ion from HM F at m /z 126 are designated by
I and H respectively.
84
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In order to assess the impact o f free glucose on the pyrolysis assay, HSA, and
HSA with addition o f 10% (w/w) glucose were pyrolyzed (Figures 6.3a and c).
Difference in intensities between glucose (1 mg) and HSA with glucose (1 mg containing
both constituents) is due to the difference in the amounts o f glucose in the two samples.
The overall fragm entation pattern o f HSA w ith glucose was a combination o f fragments
o f HSA and glucose when they were pyrolyzed separately. On the other hand, glycated
HSA gave a completely different pyrogram , generating different fragments from that o f
HSA and/or glucose upon being pyrolyzed (Figure 6.3b). Similar results were obtained
w hen pure lysozyme and lysozyme with glucose were pyrolyzed (Figures 6.3d and e).
This suggests that pyrolysis can be used as an analytical tool to distinguish proteins-sugar
m ixtures from glycated proteins. The corresponding Extracted Ion chrom atograms for
m /z 144 are shown in Figure 6.4. Note that glycated HSA, HSA w ith glucose, lysozyme
w ith glucose and glucose alone all generated the M + ion fragment with m /z 144 from the
decomposition o f the sugar moieties having a retention time at exactly 26.29 min (±0.06
min). All sugar moieties generate the same fragm ent (M+ ion at m /z 144) at the same
retention time, suggesting that pyrolysis can be used as a fast analytical tool to
distinguish glycated proteins from unglycated proteins and detect the presence o f
glycated proteins, provided that the samples have been properly dialyzed to rem ove any
unreacted sugar and freeze-dried.
In addition, as shown in Figure 6.2, pyrolysis o f glucose alone generated four fold
excess o f HMF relative to the glycation m arker with M + at m/z 144. Pyrograms o f HSA
and lysozyme that contained glucose also contained HMF, but was absent in GHSA
85
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(Figures 6.4b, c and d). This suggests that HMF could be used as an indicator for the
presence o f free glucose. However, further work is required to validate and correlate this
assumption w ith the amount o f glycated proteins, pure proteins and free glucose that
could be present.
6.3.2
ESTABILSHING THE LIMIT O F D E T E C T IO N OF MARKER IO N S FOR
G L Y C A T E D HUMAN SE R U M A LB U M IN
In determ ining the limit o f detection for detecting glycation o f commercially
available glycated protein, GHSA, the presence o f the diagnostic m arker compound 2,3dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (M+ at m/z 144) was investigated. This
ion was absent in pyrogram o f pure HSA (Figure 6.5). Upon pyrolyzing 0.1918 m g to
1.4074 mg o f GHSA, the m arker compound only appeared in the highest concentration
(Table 6.3). The corresponding signal intensities o f the duplicates generated from
deconvoluting the peak was 0.00697% and 0.00768% relative to the integrated TIC (total
ion current) o f the whole chromatogram, this correlates to the presence 5.76 x I0 '8 moles
o f hexose based on 2.7 m oles o f hexose/m ole o f protein found in the starting GHSA. In
other words, the limit o f detection o f GHSA was found to be 1.4074 mg. Future work on
pyrolyzing amounts o f G HSA slightly higher and lower than 1.4074 mg can be done so
as to generate a calibration equation for GHSA.
6.3.3
PREDICTION OF THE EXTENT OF G L Y C A T IO N IN G L Y C A T E D
LY SO Z Y M E WITH PYROLYSIS ASSAY
Figure 6.6 shows the TIC o f the glycated lysozymes at different incubation
periods. The overall degradation pattern o f the lysozyme rem ained constant with m inimal
86
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
difference as the incubation period increased. W hen only the diagnostic ion at m/z 144
was extracted from the pyrograms, the intensity o f the peak at 26.29 m in increased from
30 kCounts for 2-day incubation to more than 300 kCounts for 14-day incubation (Figure
6.7). Samples were dialyzed and freeze-dried to remove any unreacted sugar, so the
intensity o f the signal solely referred to the sugar moieties that were attached to the
protein upon incubation. This suggests that increasing the incubation period increased the
amount o f sugar m oieties attached to the protein, and this is in agreement with the
increase in the average num ber o f sugar molecules attached per m olecule o f lysozyme
(Yeboah et al., 2000). Quantitating the signal intensity o f the deconvoluted peaks at
retention time o f 26.29 min, a linear correlation betw een the signal intensity at m/z 144
with the duration o f incubation was obtained (Figure 6.8). In addition, w hen the signal
intensity was correlated with the average sugar loading values o f the lysozyme reported
in literature (determ ined by ESI-M S analysis), a linear correlation was generated as well,
which can be used to track and predict the average sugar loads o f any glycated lysozyme
prepared with the same procedure (Figure 6.9).
6.3.4
IN T R O D U C T IO N T O T H E C O N C E P T O F S IM IL A R IT Y IN D EX TO
ASSESS ST R U C T U R A L C H A N G E S IN P R O C E S S E D P R O T E IN S
Lysozyme/glucose m ixtures that had been subjected to 8 hours o f HHP and 10
minutes o f microwave irradiation were pyrolyzed after the freeze-drying step. By visual
examination o f the pyrogram s generated, the two pyrogram s seem ed to show similar
num ber o f peaks with minimal difference in their intensities (Figure 6.10). Fluorescamine
assay however, indicated that lysozyme sample subjected to 8 hours o f HHP generated 2
times as much glycation as the sample that was subjected to 10 m inutes o f microwave
87
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
irradiation. To evaluate the differences between these two samples more accurately, their
total ion chrom atogram s were compared with the total ion chrom atogram o f a model
reference system containing lysozyme and glucose. Spectral comparison parameters % Purity and % Fit values - were generated by the SATURN software and listed in Table
6.4. "% Purity" refers to the difference in the num ber o f peaks generated between the
tested sample and the reference, and "%Fit" refers to how similar the same peaks that are
comm on in both the reference and the tested sample are in terms o f their intensity. Since
both param eters are equally important to assess the differences betw een the two samples,
w e introduced the concept o f "Similarity Index" (SI) by adding the two values and
dividing by 2, the obtained values for different systems are listed in Table 6.4.
Inspection
o f Table
6.4
indicates
that
when
the
reference
system
is
lysozym e/glucose (R 1), the similarity index (SI) o f the m icrowave treated sample
(SI=88.2) is higher than that o f HHP treated sample (SI=76.5). This is consistent with the
glycation values with the HHP sample having more glycation determined with the
fluorescamine assay. A high similarity index to that o f lysozym e/glucose reference
system indicates presence o f more unreacted protein and sugar in the test sample and a
lower SI value indicates the presence o f less free sugar and protein, and more glycated
lysozyme is in the test sample. As expected, the SI values for pure lysozyme (SI=62.6),
pure glycated lysozyme LG-2 (81=59.1) and pure glucose (SI=62.4) were much lower as
shown in Table 6.4, due to the difference in structures o f the samples. It is expected
therefore when the reference system is changed into glycated lysozym e (R2), the SI
values will change such that the sample with higher glycation (HHP treated) will be more
88
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
sim ilar to the reference since it had more glycation as determined by the fluorescamine
assay. Inspection o f the values reported in Table 6.4 confirms this prediction. In addition,
the sim ilarity indices reported in Table 6.4 for LG-5, LG-10 and LG-14 glycated samples
relative to LG -2 can be used to predict the differences or similarities in their relative
functional properties.
6.4
C O N C L U S IO N
T he feasibility o f using Pyrolysis-GC/M S as an analytical technique to detect
glycation was tested. Free glucose in the reaction system and bound glucose as ARP
generated different m arkers upon pyrolysis, which can be used as a tool to distinguish
unglycated proteins from glycated proteins. Attem pt to generate a universal calibration
based on the intensity o f the M + ion at m/z 144 for glycated proteins was not possible,
since different proteins have different efficiencies to generate the m arker due to
differences in their size and glycation sites. Generation o f a calibration for glycation
therefore is protein specific. The limit o f detection o f the diagnostic ion at m/z 144 for
GHSA which has 2.7 moles o f hexose per m ole o f protein and a 66,000 Da molecular
weight was found to be 1.4074 mg. Lysozyme samples with different degrees o f
glycation produced a linear response with the signal intensity o f the diagnostic ion at m/z
144. In addition, the subsequent sugar loading values o f the glycated lysozyme also
correlated linearly with the signal intensity produced by the m arker. Finally, the
introduction o f the concept o f similarity index based on total ion current o f pyrograms
can help to predict the changes in the structures o f the glycated proteins and hence the
subsequent functional properties.
89
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kCounts j
DIAGNOSTIC ION AT
A
M/Z 144
1.50-
1.25-
1. 0 0 -
GHSA
HSA
0.25-
o.oo25.0
26.0
26.1
26.1
m inutes
Figure 6.5
Overlaid chromatograms o f HSA and GHSA.
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R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
k C ounts-
700600500400300-
200
100
-
-
kC o u n ts
700-
600
-
500400300-
200
-
100-
MA
kCounts.
700600*
500400300200
100-
k C o u n ts.
700600500400300'
200
100
-
iilil
-
20
40
m in u tes
Figure 6.6
Pyrogram s showing the total ion current o f the lysozym e-glucose
incubated samples for (a) 2 days; (b) 5 days; (c) 10 days; (d) 14 days. Peaks
containing the M+ ion at m/z 144 are designated by I.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
kCounts
70
60
(a)
50
40
30
20
10
0
kCounts
70
60
50
40
30
(b)
20
10
0
kCounts
150
125
(c)
100
75
50
25
0
kCounts
300
(d )
250
200
150
100
50
0
10
20
30
40
minutes
Figure 6.7
Extracted Ion chrom atograms (m/z 144) o f the lysozyme-glucose
samples being incubated for (a) 2 days; (b) 5 days; (c) 10 days; (d) 14 days. Peaks
containing the M + ion at m/z 144 is designated by I.
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R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
2.5 -
0.1869X
0.9707
0
2
4
6
8
12
10
14
Days under re g u la r In c u b a tio n
Figure 6.8
Correlation o f the tim e o f the lysozym e samples being incubated at 50°C
over a period o f 14 days versus the signal intensity o f the diagnostic peak after
deconvolution.
2.5
y = 0.2686X
R2 = 0.962
o>
0.5
0
2
6
4
8
10
A v e ra g e n u m b e r o f s u g a r m o le c u le s per m o le c u le of
lysozym e
Figure 6.9
Correlation o f the average num ber o f sugar m olecules attached per
molecule of lysozyme versus the signal intensity o f the diagnostic peak after
deconvolution.
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M C ounts
(a)
MCounts
4
(b)
H
f
I
.-t'--.'V~U. ,.,1_
10
20
30
40
minutes
Figure 6.10
Pyrograms showing the total ion current o f the lysozyme samples
that had been subjected to (a) 8 hours o f HHP and (b) 10 m inutes o f focused
microwave irradiation. Peaks containing the M+ ion at m/z 144 and the ion from
HMF at m/z 126 are designated by I and H respectively.
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Table 6.3
Amount o f GHSA pyrolyzed and the corresponding signal intensity o f the
diagnostic p e a k after deconvolution
Signal intensity (%)
First o f the duplicate
Second of the duplicate
.. (m B)
... - ..
0
0.1918
0
0
0
0.3636
0
0
0.4043
0
0
0.6190
0
0
0.7955
1.2075
0
0
1.47074
0.006971
0.007681
Am ount o f deconvoluted component (2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran4-one ) relative to the integrated total ion count for the entire chromatogram
A m ount o f GHSA
Table 6.4
Comparison o f treated lysozyme samples with lysozyme/glucose based on
% purity and % fit, and the corresponding Sim ilarity Index (Si)._________ _______________
Item being
% Purity
Reference (R)
% Fit
SI
compared with R
Rl
100
100
100
MW2
Lysozyme +
85.5
90.9
88.2
glucose
H H PJ
71.3
81.7
76.5
(R l)
Glucose
57.7
67.1
62.4
LG-2
53.7
64.4
59.1
Lysozyme
47.5
77.7
62.6
R2
100
100
100
MW2
52.3
61.6
57.0
HH PJ
60.8
73.4
67.1
LG-2
Lysozyme
62.5
76.6
69.6
(R2)
LG-5
87.6
92.6
90.1
LG -10
79.5
88.8
84.2
LG-14
55.2
65.2
60.2
similarity index = (%Purity + %Fit)/2)
2lysozyme sample subjected to 10 min o f m icrowave irradiation
J lysozyme sample subjected to 8 hours o f HHP
95
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7
GENERAL CONCLUSION
Generation o f glycated proteins with high energy exposure was investigated in
this study. Lysozym e was found to have its extent o f glycation accelerated w ith the
application o f HHP and focused microwave irradiation. BSA, on the other hand, resulted
in lower extent o f glycation under both treatments as compared to regular incubation.
Glycated B SA m ight have undergone reversible reactions followed by the formation o f
crosslinks resulting in extensive denaturation to the protein w ith lim ited glycation.
A pplication o f the high energy exposures can therefore, be beneficial or detrimental to
the protein depending on the nature and size o f the specific proteins under investigation.
Pyrolysis-GC/M S analysis on the other hand, serves as a rapid and reproducible
technique to assay glycated proteins, using the diagnostic m arker compounds generated
through pyrolysis. Similarities betw een pyrogram s can be assessed using the concept of
sim ilarity index, to determine the extent o f structural changes in the glycated proteins.
Future work to be done include more detailed calculations o f the protein content
and the extent o f glycation. Processing time under HHP and focused microwave
irradiation on the protein-sugar m ixtures can be shortened so as to better detect the
optim um extent o f glycation with the least damage to the proteins. Validation on the
assumptions drawn on pyrolysis should be perform ed, so that it could be used as a fast
technique in determining the extent o f glycation o f proteins.
96
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A P P E N D IC E S
A1
T A B L E S O F RAW DATA
Raw data obtained from Lowry tests and fluorescamine assays o f the different
treatm ents are tabulated in this section.
A l.l
LOWRY T E S T O F LYSOZYME SA M PLES
Table A l . l
Absorbance at 540 nm o f the lysozyme incubation samples with no
sodium azide
A b s o rb a n c e a t 5 4 0 nm
Sample
1 st
2nd
3 rd
0 .6 9 0
0-1
0 .6 8 1
0 .6 8 9
0 -2
0 .7 3 9
0 .7 5 2
0 .7 5 5
0 -3
0 .7 8 5
0 .7 9 5
0 .7 9 6
0 .2 6 4
6-1
0 .2 5 8
0 .2 6 1
6 -2
0 .2 6 0
0 .2 6 3
0 .2 6 7
6 -3
0 .2 4 8
0 .2 5 4
0 .2 5 5
8-1
0 .2 9 8
0 .3 0 4
0 .3 0 6
8 -2
0 .2 8 2
0 .2 8 9
0 .2 9 0
8 -3
0 .2 8 0
0 .2 8 7
0 .2 8 8
10-1
0 .2 8 2
0 .2 8 8
0 .2 9 0
1 0 -2
0 .2 7 7
0 .2 8 4
0 .2 8 7
1 0 -3
0 .2 6 9
0 .2 7 3
0 .2 7 7
1 2-1
0 .2 9 7
0 .3 0 2
0 .3 0 5
1 2 -2
0 .3 0 9
0 .3 1 4
0 .3 2 1
1 2 -3
0 .2 9 1
0 .2 9 5
0 .3 0 4
c6-1
0 .2 6 9
0 .2 7 6
0 .2 7 3
c 6 -2
0 .2 8 2
0 .2 8 5
0 .2 8 4
c 6 -3
0 .2 7 9
0 .2 6 7
0 .2 6 5
c8-1
0 .2 2 4
0 .2 3 9
0 .2 3 7
c 8 -2
0 .2 4 7
0 .2 4 8
0 .2 4 4
c 8 -3
0 .2 6 2
0 .2 6 3
0 .2 6 3
C 10-1
0 .2 3 0
0 .2 3 0
0 .2 2 6
c10-2
0 .2 3 1
0 .2 3 1
0 .2 3 1
C 1 0 -3
0 .2 2 8
0 .2 2 9
0 .2 3 0
c 1 2-1
0 .2 2 0
0 .2 1 8
0 .2 1 5
C12-2
0 .2 2 2
0 .2 2 4
0 .2 2 3
C 1 2 -3
0 .2 2 5
0 .2 2 2
0 .2 2 3
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without permission.
Table A1.2
Absorbance at 540 ran of the lysozyme incubation samples with 0.01%
sodium azide
Sample
Absorbance at 540 nm
2nd
0 -3
1st
0.862
0.888
0.814
2-1
0 .8 5 8
0 .8 7 4
0 .8 7 7
2 -2
0 .8 3 9
0 .8 6 3
0 .8 6 8
2 -3
0 .8 5 6
0 .8 7 2
0 .8 7 4
6-1
0 .8 5 9
0 .8 8 0
0 .8 8 1
6-2
0 .8 1 3
0 .8 1 3
6 -3
0.794
0.852
0.871
0 .8 7 8
8-1
0 .8 2 9
0 .8 4 0
0 .8 4 4
8-2
8-3
10-1
0 .8 5 6
0 .8 7 3
0 .8 7 7
0 .7 9 5
0 .8 0 2
0 .8 0 3
0 .8 4 3
0 .8 6 3
0 .8 6 4
1 0 -2
0 .8 2 2
0 .8 3 7
0 .8 3 8
1 0 -3
0 .8 3 6
0 .8 5 4
0 .8 5 6
12-1
12-2
0 .7 6 7
0 .7 6 7
0 .9 0 8
0 .9 0 7
0.840
0 .8 4 1
c0-1
0.759
0.890
0.819
0.913
0 .9 1 S
0 .9 2 3
cO -2
0 .9 8 1
0 .9 9 5
0 .9 9 6
cO -3
0 .8 9 3
0.871
0.773
0.855
0.855
0.804
0.930
0.925
0.949
0.934
0.900
0.879
0 .9 0 1
c2-1
0 .7 7 5
0 .7 7 5
0-1
0-2
1 2 -3
c2-2
c 2 -3
c6-1
c 6 -2
3rd
0 .8 7 9
0 .8 8 2
0.907
0.832
0 .9 1 3
0 .8 3 4
0 .8 8 1
0 .8 6 6
0 .8 6 6
0 .8 6 7
0 .8 7 1
0 .8 1 0
0 .8 1 2
0 .9 3 7
0 .9 3 9
0 .9 3 1
0 .9 3 2
0 .9 5 3
0 .9 5 6
0 .9 4 0
0 .9 4 0
0.902
0 .9 1 3
0 .9 1 2
0 .8 6 8
0 .8 6 8
0 .9 0 4
0 .9 0 5
C12-1
c12-2
0.859
0.895
0.951
0.847
C12-3
0 .8 4 2
c 6 -3
c8-1
c 8 -2
c8-3
d0-1
dO-2
dO-3
0 .9 6 0
0 .9 6 1
0 .8 5 1
0 .8 5 1
0 .8 5 6
0 .8 5 8
98
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Table A1.3
Absorbance at 540 nm of the lysozyme samples that have been subjected
to HHP at 400 MPa
Sample
A b s o rb a n c e a t
2nd
0.586
0.538
0.545
0.551
0.548
0.541
0.561
0.544
0.533
0.602
0.595
0.584
0.627
0.597
0.585
0.606
0.579
0.582
1st
8-1
8-2
8-3
24-1
24-2
24-3
48-1
48-2
48-3
c 8 -1
c8-2
c8-3
C24-1
C24-2
C24-3
C48-1
C48-2
C48-3
0.585
0.531
0.534
0.540
0.541
0.530
0.559
0.540
0.528
0.592
0.586
0.574
0.617
0.586
0.575
0.599
0.572
0.572
540 nm
3rd
0.588
0.539
0.547
0.552
0.549
0.543
0.561
0.545
0.533
0.603
0.595
0.584
0.630
0.602
0.596
0.609
0.580
0.586
99
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Table A1.4
Absorbance at 540 nm of the lysozyme samples that have been subjected
to microwave irradiation from 10 minutes to 60 minutes
Sample
A b s o rb a n c e a t 5 4 0 nm
1st
2nd
3rd
1 0-1
0 .3 3 4
0 .3 4 5
0 .3 4 7
1 0 -2
0 .3 7 4
0 .3 8 9
0 .3 9 2
10-3
0.351
0 .3 6 8
0 .3 7 2
2 0 -1
0 .3 8 2
0 .3 9 8
0 .4 0 2
2 0 -2
0 .4 4 4
0 .4 5 4
0 .4 5 7
20-3
30-1
0.464
0 .4 8 4
0 .4 8 8
0 .4 2 4
0 .4 4 2
0 .4 4 8
3 0 -2
0 .4 8 1
0 .5 0 1
0 .5 0 6
30-3
40-1
0 .4 4 1
0 .4 5 0
0 .4 5 1
0 .3 8 9
0 .4 1 4
0 .4 2 1
4 0 -2
0 .3 6 3
0 .3 9 0
0 .4 0 0
4 0 -3
0 .4 2 7
0 .4 4 6
0 .4 5 1
50-1
50-2
0 .3 9 1
0 .4 1 0
0 .4 1 5
0 .4 3 2
0 .4 5 1
0 .4 5 6
5 0 -3
0 .4 3 4
0 .4 5 4
0 .4 5 9
6 0 -1
0 .4 3 3
0 .4 5 1
0 .4 5 9
60-2
0 .4 1 0
0 .4 2 9
0 .4 3 8
6 0 -3
0 .4 4 1
0 .4 5 6
0 .4 6 1
C 1 0 -1
0 .4 7 3
0 .4 8 7
0 .4 9 2
C 1 0 -2
0 .5 1 2
0 .5 2 6
0 .5 3 2
C 1 0 -3
0 .5 1 5
0 .5 4 0
0 .5 4 8
C 20-1
0 .4 4 7
0 .4 5 6
0 .4 5 9
C 2 0 -2
0 .5 5 9
0 .5 6 8
0 .5 7 2
C 2 0 -3
0 .5 4 4
0 .5 5 7
0 .5 6 1
C 30-1
0 .4 3 4
0 .4 5 9
0 .4 6 9
C 3 0 -2
0 .4 5 4
0 .4 6 7
0 .4 7 2
C 3 0 -3
0 .4 6 7
0 .4 7 9
0 .4 8 4
C 4 0 -1
0 .4 5 5
0 .4 7 5
0 .4 8 2
C 4 0 -2
0 .2 8 6
0 .2 9 5
0 .2 9 5
C 4 0 -3
0 .5 0 2
0 .5 1 9
0 .5 2 9
C 5 0 -1
0 .4 6 6
0 .4 8 3
0 .4 9 4
C 5 0 -2
0 .5 3 3
0 .5 5 4
0 .5 5 7
C 5 0 -3
0 .4 5 8
0 .4 6 7
0 .4 7 3
C 6 0 -1
0 .6 0 1
0 .6 2 8
0 .6 3 9
C 6 0 -2
0 .5 3 9
0 .5 6 0
0 .5 7 0
C60-3
0 .4 6 0
0 .4 6 9
0 .4 7 4
■
100
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A1.2
F L U 0M E SC A M 1N E ASSAY O F L Y SO Z Y M E SA M PLES
Table A 1.5
azide added
Relative fluorescence o f the lysozyme incubation samples w ith no sodium
Sample
0-1
0-2
0-3
6-1
6-2
6 -3
Relative fluorescence
@ m in 3
@ min 1
1 0 0 .3
93.3
@ min
5
1 0 1 .9
8 8 .3
9 4 .2
9 5 .1
97.4
15.0
16.7
1 0 4 .2
1 0 3 .1
1 5 .7
1 5 .7
1 7 .3
1 7 .8
1 5 .3
1 5 .7
1 5 .7
8-1
1 8 .9
1 9 .9
1 9 .8
8 -2
20.0
2 0 .6
2 0 .4
8-3
1 9 .0
1 9 .3
1 9 .4
1 0-1
15.9
1 6 .5
1 6 .3
10-2
10-3
12-1
1 3 .6
1 4 .0
1 3 .7
1 8 .7
1 8 .2
1 8 .9
1 8 .6
1 8 .7
1 2 -2
18.7
17.8
1 8 .4
1 8 .4
1 2 -3
1 6 .5
17.1
1 7 .0
c 6 -1
3 0 .2
3 1 .5
3 1 .0
c 6 -2
2 9 .0
3 0 .6
3 0 .0
c 6 -3
27.1
2 8 .4
2 7 .6
c8-1
c8-2
2 7 .1
2 8 .0
2 7 .8
2 6 .9
2 8 .1
2 7 .2
c 8 -3
2 5 .5
2 6 .6
2 6 .4
C 10-1
2 5 .1
2 5 .7
2 5 .5
ctO-2
c10-3
2 4 .9
2 5 .7
2 5 .5
2 3 .7
2 3 .8
2 3 .3
C 12-1
2 0 .1
2 1 .3
2 0 .5
C 1 2 -2
1 9 .9
2 0 .3
2 0 .0
C 1 2 -3
2 0 .1
2 0 .5
1 9 .9
101
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Table A l.6
Relative fluorescence of the lysozyme incubation samples with 0.01%
sodium azide
Sample
0-1
0 -2
0-3
2-1
2-2
2-3
6-1
6-2
6-3
8-1
Relative fluorescence
@ min 3
@ min 1
58.7
55.5
68.0
63.0
60.7
56.9
6 5 .0
61.7
63.1
59.5
47.0
44.7
58.7
5 5 .6
58.4
5 5 .4
5 8 .3
55.8
@ min 5
58.5
68.0
59.5
64.9
6 3 .2
4 6 .1
5 8 .1
5 7 .6
5 8 .3
6 2 .1
5 8 .8
6 3 .1
55.1
56.9
5 6 .8
8 -3
5 0 .2
5 2 .5
5 2 .3
10-1
10-2
10-3
12-1
12-2
12-3
c0-1
5 7 .5
6 0 .2
5 9 .6
5 4 .9
5 8 .6
5 6 .7
5 3 .4
4 4 .1
56.2
46.7
4 6 .4
6 0 .9
6 4 .8
6 4 .9
5 5 .3
5 7 .0
6 0 .1
58.6
62.5
6 2 .2
CO-2
6 3 .6
6 7 .1
6 8 .3
c0-3
57.2
6 1 .8
6 1 .7
C2-1
5 8 .5
6 1 .6
c2-2
5 2 .3
C 2 -3
61.7
59.2
56.2
61.8
61.9
51.8
61.8
53.9
64.9
62.2
8 -2
c6-1
c 6 -2
c 6 -3
c8-1
c 8 -2
c8-3
c10-1
dO-2
ciO-3
cl 2-1
C12-2
C 1 2 -3
5 5 .5
5 9 .4
5 6 .0
58.6
61.5
53.3
56.0
5 5 .7
5 3 .1
6 2 .4
5 9 .3
5 8 .4
5 8 .9
65.3
65.3
53.5
57.5
62.8
59.3
61.0
64.4
55.6
58.4
6 2 .9
6 4 .6
5 5 .6
5 8 .2
6 2 .9
5 8 .0
5 7 .8
6 5 .1
5 5 .3
5 8 .8
102
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Table A l.7
Relative fluorescence of the lysozyme samples being subjected to HHP
Sample
8-1
8-2
8-3
2 4 -1
24-2
24-3
48-1
48-2
48-3
Relative fluorescence
@ m in 3
@ min 1
37.0
36.0
37.8
35.7
36.7
35.1
36.6
34.7
@ min 5
37.6
36.4
35.7
3 5 .6
3 4 .8
3 6 ,7
3 6 .5
3 3 .6
3 5 .5
3 5 .8
3 9 .2
4 0 .6
3 9 .2
3 7 .5
3 9 .6
3 8 .8
3 5 .2
3 7 .0
c 8 -3
42.0
41.3
40.8
C24-1
4 5 .2
37.2
44.6
44.0
43.6
47.6
C 2 4 -2
4 1 .7
4 4 .0
4 2 .9
C 2 4 -3
40.6
4 2 .0
4 1 .2
c 4 8 -1
3 8 .7
4 0 .5
3 9 .5
C48-2
C48-3
38.1
4 0 .9
3 9 .0
3 2 .6
3 4 .7
3 4 .1
c 8 -1
c 8 -2
4 3 .9
4 4 .2
4 3 .8
4 7 .1
103
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Table A1.8
Relative fluorescence of the lysozyme samples that have been subjected to
microwave irradiation
Sam ple
10-1
10-2
10-3
20-1
R elative flu orescen ce
@ m in 3
@ min 1
@ min 5
1 8 .6
1 9 .5
1 9 .2
2 0 .1
2 0 .9
2 0 .6
1 8 .4
1 9 .2
1 8 .8
2 1 .3
2 2 .0
2 2 .1
2 0 -2
2 1 .1
2 2 .3
2 1 .8
20-3
30-1
30-2
2 4 .4
2 3 .5
2 3 .3
1 4 .8
15.1
1 4 .9
2 6 .7
2 7 .8
2 7 .7
3 0 -3
2 2 .7
2 4 .4
2 4 .1
40-1
40-2
40-3
50-1
2 2 .5
2 4 .2
2 3 .3
2 2 .6
2 3 .8
2 3 .0
2 2 .0
2 3 .0
2 3 .2
2 1 .2
2 1 .4
2 1 .7
5 0 -2
2 1 .4
2 1 .8
2 1 .9
5 0 -3
2 1 .8
2 2 .5
2 2 .3
6 0 -1
2 5 .7
2 6 .3
2 6 .4
6 0 -2
2 2 .5
2 3 .7
2 3 .4
6 0 -3
2 0 .0
2 0 .6
2 1 .0
C 1 0 -1
2 4 .7
2 6 .2
2 5 .4
C10-2
2 9 .5
2 9 .9
2 9 .5
C 1 0 -3
2 8 .7
2 8 .8
2 9 .5
C 2 0 -1
2 4 .6
2 4 .9
2 5 .1
C 2 0 -2
3 1 .3
3 2 .7
3 1 .4
C20-3
3 3 .9
3 4 .5
3 4 .6
C 3 0 -1
2 2 .3
2 3 .2
2 3 .3
C 3 0 -2
2 0 .6
2 2 .6
2 2 .6
C 3 0 -3
2 3 .7
2 4 .5
2 4 .4
C 4 0 -1
2 1 .7
2 1 .9
2 1 .2
C 4 0 -2
1 3 .2
1 3 .7
1 3 .6
C 4 0 -3
3 2 .6
3 3 .1
3 3 .3
C 5 0 -1
2 7 .1
2 8 .4
2 7 .9
C 5 0 -2
3 1 .8
3 2 .3
3 2 .3
C50-3
2 4 .7
2 5 .7
2 5 .5
C 6 0 -1
3 6 .1
3 8 .8
3 7 .5
C 6 0 -2
3 1 .1
3 2 .4
3 2 .1
C 6 0 -3
2 3 .7
2 4 .5
2 3 .8
104
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A2
T A B L E S OF RAW D A TA OF BSA
A2.1
L O W R Y T E S T O F BSA SA M PLES
Table A2.1
Absorbance o f the BSA incubation samples from 0 to 12 days at 540
Absorbance at 540 nm
Sam ple
1st
2n d
3 rd
0-1
0 .4 4 9
0 .4 6 1
0 .4 6 2
0 -2
0 .4 3 5
0 .4 5 1
0 .4 5 3
0 -3
0 .4 4 1
0 .4 5 3
0.44
6-1
0 .3 9 7
0 .4 0 7
0 .4 1 0
6 -2
0 .4 3 8
0 .4 3 8
0 .4 4 8
6 -3
0 .4 2 1
0 .4 2 9
0 .4 3 2
8-1
0 .4 2 2
0 .4 3 0
0 .4 3 0
8 -2
0 .3 9 7
0 .4 0 4
0 .4 1 0
8 -3
0 .4 3 6
0 .4 4 4
0 .4 4 8
10-1
0 .3 7 8
0 .3 8 9
0 .3 8 9
1 0 -2
0 .4 2 3
0 .4 3 1
0 .4 3 2
1 0 -3
0 .4 2 6
0 .4 3 1
0 .4 3 0
12-1
0 .4 2 5
0 .4 2 9
0 .4 2 8
1 2 -2
0 .4 0 6
0 .4 1 0
0 .4 1 4
1 2 -3
.0 .3 4 5
0 .3 5 2
0 .3 5 8
0 .3 6 6
0 .3 7 9
0 .3 7 4
c 6 -2
0 .2 7 2
0 .2 7 2
0 .2 7 2
c 6 -3
0 .2 8 3
0 .2 8 5
0 .2 8 7
c8-1
0 .3 8 4
0 .3 8 9
0 .3 9 1
c 8 -2
0 .3 9 5
0 .3 9 9
0 .3 9 9
c 8 -3
0 .2 7 7
0 .2 8 3
0 .2 8 2
c 1 0 -1
0 .4 4 3
0 .4 4 1
0 .4 4 1
C 1 0 -2
0 .3 8 6
0 .3 9 2
0 .3 9 3
dO-3
0 .2 7 6
0 .2 8 0
0 .2 8 0
C 12-1
0 .4 2 1
0 .4 2 5
0 .4 2 4
C 1 2 -2
0 .3 8 5
0 .3 8 9
0 .3 9 0
C12-3
0 .2 7 4
0 .2 7 7
0 .2 6 9
c6-1
105
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Table A2.2
Absorbance at 540 nm of the BSA samples being subjected to HHP
Sample
8-1
8-2
8-3
1st
1.049
0.256
Absorbance a t 5 4 0 nrn
2 nd
3 rd
1 .1 1 5
1.120
0 .2 7 7
0 .2 8 6
0 .5 6 5
0 .6 1 3
0 .6 3 5
2 4 -1
0 .5 5 8
0 .5 9 9
0 .6 1 9
2 4 -2
0 .5 4 8
0 .5 8 7
0 .6 0 4
2 4 -3
0 .2 9 8
0 .3 2 5
0 .3 3 0
48-1
48-2
0 .3 8 9
0 .4 1 9
0 .4 3 2
0 .5 4 6
0 .5 9 2
0 .6 0 4
4 8 -3
0 .4 8 1
0 .5 1 1
0 .5 2 7
c 8 -1
0 .2 7 6
0 .2 9 0
0 .2 9 6
c 8 -2
0 .4 0 8
0 .4 2 9
0 .4 4 0
c 8 -3
0 .4 0 3
0 .4 2 3
0 .4 3 3
C24-1
0 .2 5 9
0 .2 7 1
0 .2 7 9
C24-2
0 .3 5 4
0 .3 7 4
0 .3 8 6
C 2 4 -3
0 .4 6 6
0 .4 9 0
0 .5 0 5
C48-1
0 .1 4 1
0 .1 4 6
0 .1 4 4
C48-2
C48-3
0 .2 2 9
0 .2 3 4
0 .2 4 3
0 .2 8 5
0 .2 9
0 .3 0 7
106
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Table A2.3
Absorbance at 540 ran of the BSA samples that have been subjected to
microwave irradiation
Sample
10-1
1 0 -2
10-3
20-1
2 0 -2
2 0 -3
30-1
A b s o rb a n c e a t 5 4 0 n m
1st
0.195
0.987
1.331
2nd
3rd
0 .2 0 6
0 .2 1 1
1 .0 3 9
1 .0 5 9
1 .3 8 9
1 .4 0 8
0 .9 9 0
1 .0 3 9
1 .0 5 9
0.919
1.359
0.030
0 .9 6 8
0 .9 8 6
1 .4 1 2
1 .4 2 8
0 .0 3 9
0 .0 3 7
3 0 -2
1 .0 8 2
1 .1 1 7
1 .1 2 4
30-3
40-1
40-2
40-3
50-1
0 .6 8 9
0 .7 2 3
0 .7 3 4
0 .0 1 2
0 .0 1 1
0 .8 4 7
0 .8 5 2
5 0 -2
0.015
0.822
0.878
0.018
0.840
5 0 -3
0 .2 7 0
0 .2 8 1
0 .2 8 1
60-1
60-2
0 .0 9 9
0 .1 0 6
0 .1 1 1
0 .2 1 1
0 .2 2 6
0 .2 2 6
6 0 -3
0 .8 3 2
0 .8 5 3
0 .8 7 8
C10-1
0 .0 9 0
0 .0 9 0
0 .1 2 1
0 .1 2 0
0 .4 3 7
0 .4 4 4
C 2 0 -1
0.087
0.115
0.420
0.366
0 .3 7 8
0 .3 8 5
C20-2
0 .2 1 4
0 .2 2 0
0 .2 2 2
C 2 0 -3
0 .3 5 2
0 .3 6 5
0 .3 7 0
C30-1
0.300
0 .3 0 7
0 .3 1 4
C 3 0 -2
0 .1 2 5
0 .1 2 9
0 .1 2 8
C 3 0 -3
0 .3 7 4
0 .3 8 8
0 .3 9 2
C40-1
0 .1 1 5
0 .1 1 6
0 .0 8 5
0 .0 8 1
C 5 0 -2
0.112
0.107
0.145
0.080
0.066
0 .0 7 1
0 .0 7 0
C 5 0 -3
0 .1 2 8
0 .1 3 8
0 .1 4 2
C 6 0 -1
0 .1 3 2
0 .1 3 4
0 .1 3 3
C 6 0 -2
0 .0 9 5
0 .1 0 1
0 .1 0 2
C60-3
0 .0 9 1
0 .0 9 4
0 .0 9 6
C 1 0 -2
C10-3
C 4 0 -2
C 4 0 -3
C 5 0 -1
0 .9 0 9
0 .9 2 1
0 .0 1 7
0 .0 1 7
0 .8 5 7
0 .8 6 9
0 .1 1 1
0 .1 1 0
0 .1 5 0
0 .1 4 9
107
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A2.2
F L U O R E S C A M IN E
Table A2.4
Sample
0-1
0-2
0-3
6-1
6-2
6-3
8-1
8-2
8-3
10-1
10-2
10-3
12-1
12-2
12-3
c6-1
c6-2
c6-3
c8-1
c8-2
c8-3
c10-1
c10-2
c10-3
cl 2-1
C12-2
C12-3
ASSAY OF BSA SAMPLES
Relative fluorescence o f the BSA incubation samples
Relative fluorescence
@ min 3
@ min 1
88.0
84.3
81.4
84.2
80.7
84.0
56.5
54.6
45.7
47.1
58.7
56.9
56.0
54.1
51.8
50.0
53.1
51.8
45.1
44.0
50.5
52.5
52.6
51.3
52.6
53.5
42.5
43.8
49.4
47.5
60.2
62.3
43.7
44.0
50.2
49.5
62.4
63.0
59.5
58.6
52.0
52.9
72.1
71.8
65.9
66.2
50.3
49.6
72.8
72.9
51.8
51.1
50.7
52.2
@ min 5
88.0
84.2
84.5
57.1
47.2
59.6
56.1
52.2
53.5
45.1
52.3
53.5
54.5
44.6
49.2
62.3
43.5
50.2
63.2
59.4
52.9
72.6
66.2
50.4
73.7
52.8
52.0
108
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Table A2.5
Relative fluorescence of the BSA samples being subjected to HHP
Sample
8-1
8-2
8-3
24-1
24-2
24-3
48-1
48-2
48-3
c8-1
c8-2
c8-3
C24-1
C24-2
C24-3
C48-1
C48-2
C48-3
Relative fluorescence
@ min 3
@ min 1
80.6
79.7
88.1
83.7
42.6
42.6
54.9
51.9
40.5
38.7
14.8
14.4
26.1
25.3
32.8
31.5
41.0
33.9
13.8
13.2
28.8
24.6
28.2
30.0
15.0
14.5
31.4
31.9
23.8
23.6
14.9
14.2
15.0
14.2
12.2
12.1
@ min 5
80.6
92.1
43.5
51.0
41.6
15.2
26.4
33.4
35.7
13.7
24.9
30.3
14.3
31.6
23.3
15.1
14.2
12.3
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Table A2.6
Relative fluorescence of the BSA samples that have been subjected to
microwave irradiation
Sample
10-1
10-2
10-3
20-1
20-2
20-3
30-1
30-2
30-3
40-1
40-2
40-3
50-1
50-2
50-3
60-1
60-2
60-3
C10-1
C10-2
C10-3
C20-1
C20-2
C20-3
C3Q-1
C30-2
C30-3
C40-1
C40-2
C40-3
C50-1
C50-2
C50-3
C60-1
C60-2
C60-3
Relative fluorescence
@ min 3
@ min 1
12.2
13.1
119.0
123.9
147.2
141.5
112.7
94.7
103.4
100.1
91.3
88.1
2.7
3.5
113.4
109.0
45.7
50.7
2.1
1.9
66.6
63.7
72.7
74.3
2.3
2.0
83.0
86.6
21.5
22.4
2.7
3.1
10.3
11.0
58.0
58.0
8.8
8.8
19.8
20.1
24.1
24.6
21.8
21.0
11.1
11.5
20.2
20.3
21.3
21.8
25.7
24.8
14.9
14.3
10.9
11.2
11.6
11.2
10.4
11.0
8.4
8.0
5.4
5.4
11.4
12.0
45.2
42.5
9.8
8.9
10.2
11.1
@ min 5
9.2
123.6
148.7
93.7
98.6
91.6
3.5
113.8
45.1
2.1
66.9
74.0
2.2
87.2
22.0
2.7
11.0
59.2
9.4
20.0
24.6
21.7
11.6
20.8
21.6
26.0
15.0
11.5
11.6
11.1
8.5
5.4
12.0
46.5
9.0
11.5
110
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CALCULATION
A3
SA M PLE
A3.1
AN EXAMPLE FOR CALCULATING THE AMOUNT OF BSA PRESENT
IN THE SAMPLES THAT G E L L E D
Exam ple
W eight o f gel o f 24-1 BSA high pressure treated sample is 0.4092 g. 6.6
m l. o f w ater and 0.5 niL o f 12% SDS were added to the gel w ith repeated vortexing and
centrifuging to have the supernatant collected for analysis. The corresponding amount o f
BSA in the gel will be calculated in ratio to the amount o f BSA in the original solution
prepared for treatm ent (5.043 g BSA and 2.518 g o f glucose in the glucose-containing
solution in 19 m L o f water).
W eight o f B SA = 5.043 g; weight o f glucose = 2.518 g
W eight o f w ater = 19 g (taken 1 ml o f water = 1 g o f water)
Total w eight o f the original mixture = 26.561 g
Am ount o f BSA in the gel = (0.4092 g*5.043 g) / 26.561 g = 0.0777 g
The gel dissolved in 6.6 mL o f water and 0.5 mL o f 12% SDS, and 30 pL o f the solution
was used for test.
Therefore, the am ount o f BSA present in the test solution (96% purity of the BSA purchased
from Sigma)
= 0.0777 g / (6.6+5.5 mL) * 1000*0.96 * 0.03 mL = 0.3151 mg
The same calculation applied to the m icrowave samples, substituting the w eight o f the gel
with the dilutions done to the original concentration prior to being irradiated.
A3.2
1.
STEPS IN CALCULATING THE EXTENT OF GLYCATION
First, norm alize all the protein amounts to 0.1 mg for all the samples, to get the
corresponding absorbance o f the samples in the Low ry test.
I ll
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2.
T ak in g
the absorbance o f day 0 glucose-containing samples as 100%, calculate
the respective % o f protein content for all the other samples and the corresponding
amount o f p rotein in mg present in the test solution.
3.
N orm alize all the fluorescence readings to 0.1 m g o f protein with respect to the
amount o f protein present in the test solution.
4.
A ssum ing that the day 0 did not undergo any dexiaturation upon preparation, its
m easured fluorescence is taken to be the expected fluorescence at day 0.
5.
C alculate the expected fluorescence o f the other samples w ith respect to the
amount o f protein o f that test solution calculated from Low ry test and the expected
fluorescence at day 0.
6.
Calculate the difference in fluorescence by subtracting the m easured fluorescence
o f the test solution from the expected fluorescence calculated.
7.
Calculate the num ber o f lysyl groups present in the day 0 test solution.
8.
Then, calculate the num ber o f lysyl groups that got glycated or denatured from the
difference in fluorescence.
9.
Calculate the extent o f glycation by dividing the num ber o f lysyl groups that got
glycated or denatured with the total num ber o f lysyl groups present in day 0.
A 33
1.
EQUATIONS F O R THE CALCULATIONS
Amount o f soluble protein determined from Lowry test = protein content (%) *
amount o f soluble protein o f day 0 test solution/ 100
2.
Expected fluorescence= amount o f protein determ ined from Low ry test * measure
fluorescence o f the day 0 test solution / amount o f protein o f day 0 from Lowry test
112
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3.
D ifference in fluorescence = Expected fluorescence - m easured fluorescence
4.
# lysyl groups got glycated or denatured = difference in fluorescence * # lysyl
groups o f day 0 test solution / m easured fluorescence o f day 0 test solution
5.
Extent o f glycation or denaturation = # lysyl groups got glycated/denatured/ total
# o f lysyl groups o f day 0 test solution * 100
6.
A verage # o f reacted lysyl groups = # lysyl groups got glycated or denatured /
total # o f lysyl groups in day 0 test solution * the num ber o f lysyl groups present in 1
m ole o f the native protein
A3.4
AN E X A M P L E IN C A L C U L A T IN G T H E E X T E N T O F G L Y C A T IO N
Example: 6-1 o f lysozyme sample (that contained glucose).
The norm alized absorbance is 0.350. W ith reference to the absorbance o f the day 0
sample (0.895, which is assumed to have 100% protein content), protein content o f the
day 6 sample is 0.350*100/0.895 = 39.16% and the corresponding amount of protein
present is 0.03916 mg (with reference to 0.1 mg in the day 0 sample).
N orm alized fluorescence o f day 0 sample is found to be 117.5, and that o f day 6 sample
is 21.8. Therefore, the expected fluorescence that should have with 0.03916 mg o f protein
should be 46.0 (0.03916 mg * 117.5 / 0.1 mg). The difference in fluorescence between
the m easured fluorescence o f the day 6 sample and the expected fluorescence is 24.21. In
other words, the difference in fluorescence refers to the lysyl groups that got glycated by
the glucose, and this could be used to extent o f glycation by dividing it with the original
num ber o f lysyl groups present in the day 0 sample.
113
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