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Elucidation of nonthermal effects of microwave irradiationon the unfolding pathways of β-lactoglobulin and hemoglobin

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Elucidation o f N ontherm al Effects o f M icrowave Irradiation
on th e U nfolding Pathways o f p-Lactoglobulin and
H em oglobin
By
Abdul Nasser Al-Jundi
Department o f Food Science and Agricultural Chemistry
Macdonald Campus, McGill University
Montreal (Quebec)
Canada
A Thesis Submitted to the Graduate and Post-Doctoral Studies Office in
Partial fulfillment of the requirements o f the degree o f
Master o f Science
A ugust 2004
Abdul N asser Al-Jundi, 2004
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Suggested Short Title:
Nonthermal Effects of Microwave Irradiation
ii
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ABSTRACT
In recent years there has been considerable interest in the development of
microwave-based food processing technologies. Microwave radiation is considered to
have both thermal and non-thermal effects. The thermal effects are related to the heat
generated by the absorption of microwave energy by water or by organic molecules, but
very little is known about the mechanisms involved in the putative non-thermal effects. It
has been postulated that the latter could involve a direct energy transfer from the
electromagnetic field to the vibrational modes o f macromolecules, altering their
conformation. In the present study, the non-thermal effects induced upon irradiation o f
protein solutions with microwave energy were investigated by employing Fourier
transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy to
elucidate the unfolding pathways o f hemoglobin and P-lactoglobulin (5% in D 2 O)
subjected to either microwave irradiation (2450 MHz) or conventional heating. The
microwave treatments were performed for different times and numbers o f irradiation
cycles at selected temperatures to investigate the influence o f these parameters. At 54°C,
hemoglobin aggregated faster upon microwave irradiation than with conventional heating
(water bath) under the same conditions. The a-helix and 3 1 0 -helix contents o f hemoglobin
were reduced after the microwave treatment compared to conventional heating, and the
hydrophobic interior o f the protein was more exposed to solvent in the microwaveirradiated samples. In the case o f P-lactoglobulin, higher rates o f H-D exchange occurred
during microwave irradiation of D 2 O solutions at 35°C by comparison with conventional
heating at the same temperature. At 70°C (close to the aggregation temperature), Plactoglobulin aggregated faster when exposed to microwave irradiation compared to
conventional heating. The experimental data provides evidence for the existence of a nonthermal effect o f microwave irradiation on protein unfolding. A possible mechanism may
involve the direct absorption of microwave energy by the protein backbone leading to an
increase in the inter-domain motion, which may result in the loosening o f the tertiary
structure and enhanced protein aggregation close to the denaturation temperature o f the
proteins.
111
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RESUME
II y a, un interet considerable qui se developpe actuellement pour la comprehension
des effets de 1’irradiation a haute frequence des aliments dans le monde de la transformation
alimentaire. L’irradiation a haute frequence produit des effets thermiques et non thermiques.
Les effets thermiques sont relies a la chaleur generee lors de l’absorption de l’energie par
l’eau ou par les molecules organiques Par contre, tres peu est connu a ce jour sur les
mecanismes impliques par les possibles effets non thermiques; il est suppose que ces demiers
soient les resultats d’un transfert direct d’energie du champ electromagnetique au mouvement
oscillatoire des macro molecules, influen9 ant ainsi leur conformite. Dans l’etude presente, les
effets non thermiques obtenus par l’irradiation a haute frequence ou par la chaleur
conventionnelle de solutions proteiques ont ete examines en utilisant la spectroscopie infra
rouge de Fourier, la dichromatique circulaire, et la spectroscopie fluorescente pour elucider le
modele de deploiement de la proteine (5%) d’hemoglobine et de la P-lactoglobuline
solubilisee dans le D2 O lorsque soumise a une frequence (2450 MHz). L’irradiation a haute
frequence a ete effectuee a plusieurs reprises et aussi a des temperatures preselectionnees afin
de connaitre 1’ influence de ces parametres sur le modele de deploiement de la proteine. A
54°C et dans les memes conditions, l’hemoglobine s’agglutine plus rapidement lorsque elle
est soumise a l’irradiation a haute frequence par rapport a celle soumise a un traitement
thermique conventionnel en utilisant le bain-marie. Les confirmations, a-helix et 3 10 helix, de
l’hemoglobine ont ete plus reduite apres la traitement par miro ondes par rapport au
traitement thermique conventionnel; de plus, il a ete observe que la portion hydrophobique de
l’interieur de la proteine s’est retrouvee plus exposee au solvant dans les echantillons irradies
a haute frequence. Dans le cas de la P-lactoglobuline, les solutions de D20 a 35°C ont eu de
plus haut taux d’echange H-D au cours de 1’irradiation a haute frequence par rapport a celles
traitees thermiquement par la methode conventionnelle. A 70°C, qui est la temperature pres
de 1’agglutination, la p-lactoglobuline s’agglutine plus rapidement lorsque elle est exposee a
l’irradiation a haute frequence par rapport au traitement thermique. Les donnees
experimentales tendent a montrer l’evidence de la presence d’effets non thermiques sur le
deploiement des proteines lorsque elles ont ete soumises a des traitements d’irradiation a
haute frequence. Un mecanisme possible qui implique l’absorption directe de l’energie a
frequence par la structure centrale qui a donne lieu a une augmentation de la mobilite interne
IV
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ACKNOWLEDGMENTS
I would like to express my sincere appreciation to my thesis supervisor. Dr.
Ashraf.A.Ismail. Beyond his supervisory responsibility, Dr. Ismail has been a valued
friend and mentor whose encouragement, persistence and focus throughout the course o f
this study were invaluable in the completion o f this thesis.
I am greatly indebted to Dr. Varoujan Yaylayan, who not only provided access to
the Synthewave 402 microwave system but also was always available to offer assistance
and suggestions. Your help is highly appreciated. I would also like to thank Dr.
Jacqueline Sedman for her guidance and suggestions.
Particular recognition is extended to Dr. Joanne Turnbull o f the Department of
Biochemistry o f Concordia University for providing access to the fluorescence and
circular dichroism instruments and for her assistance in the preparation o f the paper that is
presented in Chapter 5 o f this thesis.
My gratitude also extends to Dr. Jocelyn Pare and Dr. Jacqueline Belanger in
Environmental Canada who offered the MAP course in microwave.
I gratefully acknowledge McGill University for the scholarship o f excellence that
I held throughout my first year o f study.
My deep admiration goes to my colleagues and friends at McGill University and
IR group, especially Pedro, Sarah and Ahmad. Thank you Jason, Marta and Gaya.
Because of all o f you, the experience was pleasant.
My cordial appreciation and my special salute to Ken W ood You made my
experience very exceptional.
I would like to extend my thanks and gratitude to the graduate students and staff
of the Food Science Department, especially Lise and Barbara, for the friendly
environment and support they offered during the course o f my studies.
A special thank goes to Dr. Selim Kermasha and Christiane Fortier for their
friendship and for their help in translating the abstract o f this thesis to French.
Last but certainly not least; I would like to take this opportunity to express my
sincere gratitude to my family, my mother, all my brothers and sisters, and all my friends
overseas for their encouragements, love and support. You have made my studies and my
life such a wonderful experience. I love you.
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TABLE OF CONTENTS
ABSTRACT................................................................................................................................. iii
RESUME....................................................................................................................................... iv
ACKNOWLEDGEMENTS....................................................................................................... v
TABLE OF CONTENTS.......................................................................................................... vi
LIST OF T A B L E S..................................................................................................................... ix
LIST OF FIG U R E S.................................................................................................................... x
LIST OF ABBREVIATIONS.......................................................................................
xiv
CHAPTER 1 General Introduction....................................................................................... 1
CHAPTER 2 Literature Review............................................................................................. 3
2.1 Electromagnetic Radiation.................................................................................................3
2.2 Microwave.............................................................................................................................. 3
2.2.1 History of M icrowaves...............................................................................................5
2.2.2 Microwave H eating.................................................................................................... 5
2.3 Dielectric Properties of Food Materials and Electric Field Interactions................7
2.4 Microwave Effects................................................................................................................ 9
2.5 Mechanisms of Inactivation/destruction of Microorganisms by M W ................... 15
2.5.1 Thermal E ffects........................................................................................................ 15
2.5.2 Kinetic E ffects...........................................................................................................15
2.5.3 Chemical Effects....................................................................................................... 15
2.6 Microwave Effects on Protein Structure......................................................................16
2.7 Mechanisms of Nonthermal Effects............................................................................... 17
2.8 Effect of Physiochemical Conditions on Microwave T reatm ent............................21
2.9 Microwave and Human Health.......................................................................................21
R eferences...........................................................................................................................24
CHAPTER 3 Nonthermal Effects of Microwave Irradiation on the Structure of
P-lactoglobulin under Different Physicochemical C onditions............. 28
3.1 Abstract................................................................................................................................ 28
3.2 Introduction........................................................................................................................ 29
3.3 Materials and M ethods.....................................................................................................31
3.3.1 Microwave Treatments.........................................................................................31
vi
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3.3.2 Conventional Heating Treatm ents.......................................................................32
3.3.3 Fourier Transform Infrared (FTIR) Spectroscopy.........................................32
3.4 R esults................................................................................................................................... 32
3.4.1 Effect o f Microwave Irradiation on the Structure of P-lg..............................34
3.4.2 Effect of Microwave Irradiation on p-lg solutions at Different p H s........... 38
3.4.2.1 Effect of microwave irradiation on the P-lg at pH 2 .......................... 40
3.4.2.2 Effect o f microwave irradiation on the P-lg at pH 4 .......................... 41
3.4.2.3 Effect of microwave irradiation on the P-lg at pH 9 .......................... 42
3.4.3 Effect of Microwave Irradiation on the P-lg in 0.5 M N aC l.......................... 43
3.4.4 Effect of Microwave Irradiation on the p-lg in 2 M N aC l..............................44
3.4.5 Comparison between Multiple MW and C.V.H. Cycles at 75°C..................45
3.5 D iscussion............................................................................................................................ 49
3.6 Proposed M echan ism ....................................................................................................... 50
R eferences........................................................................................................................... 52
CHAPTER 4 The Unfolding Pathway of p-lg under MW /C.V.H Treatm ent.......... 56
4.1 Abstract.................................................................................................................................56
4.2 Introduction.........................................................................................................................57
4.3 Materials and M ethods..................................................................................................... 59
4.3.1 Microwave Treatments........................................................................................... 59
4.3.2 Conventional Heating T reatm ents.......................................................................60
4.3.3 Fourier Transform Infrared (FTIR) Spectroscopy.........................................60
4.3.4 KG2D Software for 2D Correlation Spectroscopic A nalysis..................... .. 61
4.4 R esults...................................................................................................................................61
4.4.1 Effect of Elevated Temperatures on the Secondary Structure of p-lg
63
4.4.2 The Effect of Temperature on p-lg and H-D Exchanged P -lg .................... 66
4.4.3 Unfolding of P-lg after pre-treatment by MW or C.V.H. till 35°C...............73
4.4 2D IR Correlation Analysis of P-lg Aggregation..................................................76
4.5 Discussion and Conclusion...............................................................................................85
R eference............................................................................................................................. 88
CHAPTER 5 Nonthermal Effects of Microwave Irradiation on Hem oglobin.......... 92
5.1 Abstract................................................................................................................................ 92
5.2 Introduction..............................................................
93
5.3 Materials and M ethods..................................................................................................... 94
5.3.1 Microwave Treatments........................................................................................... 94
5.3.2 Conventional Heating T reatm ents...................................................................... 95
5.3.3 Fourier Transform Infrared (FTIR) Spectroscopy.........................................95
5.3.4 Fluorescence Spectroscopy.....................................................................................96
5.3.5 Circular D ichroism ..................................................................................................96
•
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5.3.5.1 Far UV C D ..................................................................................................96
5.4 Results and D iscu ssion .....................................................................................................97
5.4.1 Fourier Transform Infrared Spectroscopy........................................................ 97
5.4.2 Fluorescence ...........................................................................................................101
5.4.3 Circular Dichroism C D ........................................................................................ 104
5.5 C onclusion..........................................................................................
R eferen ces.........................................................................................................................107
CHAPTER 6 General C on clu sion .....................................................................................109
v i ii
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106
LIST OF TABLES
^
Page
Table 2.1 Properties o f electromagnetic radiation
4
Table 2.2 Microwave dielectric properties o f water at indicated temperatures
8
Table 4.1 Shows the sequence o f unfolding events upon heating P-lg till 55°C
71
Table 4.2 The sequence o f unfolding events upon heating H-D exchanged p-lg
72
from 25 to 55°C in D 2 O
Table 4.3 The sequence o f unfolding o f p-lg generated from a VT-FTIR run
75
(25-55°C) of a p-lg solution heated 3 times to 35°C by microwave irradiation.
Table 4.4 Shows the sequence o f unfolding events o f P-lg after subjected to
84
microwave irradiation cycles to achieve 75 °C.
ix
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LIST OF FIGURES
Figure 2.1 Electromagnetic spectrum
Figure 2.2 Microwave heating mechanisms
Figure 3.1 The crystal structure o f a single subunit o f bovine p-lg
30
Figure 3.2 The amide I region o f the FTIR spectrum o f p-lactoglobulin in D 2 O
33
Figure 3.3 Overlaid FTIR spectra in amide I absorption region o f P-lg
34
solutions in D 2 O subjected to 1-10 cycles o f microwave irradiation at 35°C.
Figure 3.4 A plot o f the changes in the intensity o f the 1692 cm '1 band in the
35
FTIR spectra o f P-lg as a function o f increasing irradiation cycles at 35°C
Figure 3.5 A plot o f the changes in the intensity o f the 1692 cm"1 band in the
36
FTIR spectra o f P-lg as a function o f increasing heating cycles at 35 °C
Figure 3.6 A plot o f the drop in the peak height o f the 1692 cm '1band in the
37
FTIR spectrum o f P-lg in D 2 O as a function o f M W and C.V.H at 35°C.
Figure 3.7A The amide I region o f FTIR spectra o f p-lg at different pHs
38
Figure 3.7B The amide I region o f FTIR spectra o f P-lg in different pHs
39
Figure 3.8 A plot o f the change in the peak height o f the 1692 cm '1 in the FTIR
40
spectra o f P-lg at pH 2 at 35°C as a function heating cycles by MW or C.V.H.
Figure 3.9 A plot o f the change in the peak height o f the 1692 cm '1 in the FTIR
41
spectra of P-lg at pH 4 at 35°C as a function heating cycles by MW or C.V.H.
Figure 3.10 A plot o f the change in the peak height o f the 1692 cm '1 in the FTIR
42
spectra of P-lg at pH 9 at 35°C as a function heating cycles by MW or C.V.H
Figure 3.11 A plot o f the change in the peak height o f the 1692 cm '1 in the FTIR
spectra o f P-lg in 0.5M NaCl at 35°C as a function heating cycles by MW or C.V.H.
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43
Figure 3.12 A plot o f the change in the peak height o f the 1692 cm '1 in the FTIR
spectra o f P-lg in 2 M NaCl at 35°C as a function heating cycles by MW or C.V.H.
Figure 3.13 Overlaid FTIR spectra in the amide I’ absorption region o f P-lg
solutions subjected to microwave irradiation to 75°C for 1, 3, 5, 7, and 10 cycles.
Figure 3.14 Overlaid FTIR spectra in the amide I’ absorption region o f P-lg
solutions subjected to conventional heating to 75°C for 1, 3, 5, 7, and 10 cycles.
Figure 3.15 A Plot o f the change in the peak height o f the 1615 cm '1 in the FTIR
spectra o f P-lg in at heat to 75°C as a function heating cycles by MW and C.V.H.
Figure 4.1 Dimer interface o f B-LG from the X-ray coordinates, 1BEB.
Figure 4.2 The 2D correlation procedure
Figure 4.3 The amide I absorption region o f the FTIR spectrum o f P-lg
Figure 4.4A The amide I band in the spectra o f P-lg in the presence o f the
1692 cm '1 band, upon heating conventionally in the FTIR cell till 55°C
Figure 4.4B The amide I band in the spectra o f P-lg (5 % w/v in D 2 O) o f H-D
exchanges P-lg upon heating conventionally in the FTIR cell till 55°C.
Figure 4.5A Superimposed difference FTIR spectra o f P-lg in D 2 O as a function
of increasing temperature, after subtraction o f the spectrum recorded at 25°C.
Figure 4.5B Superimposed difference FTIR spectra o f fully H-D exchanged
P-lg as a function o f increasing temperature.
Figure 4.6A The synchronous 2D IR spectra generated from the difference
spectra plotted in Figure 4.5A.
Figure 4.6B The asynchronous 2D IR spectra generated from the difference
spectra plotted in Figure 4.5A
Figure 4.7A The synchronous 2D IR spectra generated from the difference
spectra plotted in Figure 4.5B.
Figure 4.7B The asynchronous 2D IR spectra generated from the difference
spectra plotted in Figure 4.5B.
Figure 4.8 The overlaid deconvolved o f the VT-FTIR spectra o f P-lg solution
exposure to 3 cycles o f microwave irradiation.
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Figure 4.9A The synchronous map generated from a VT-FTIR run (25-55 °C)
74
of a P-lg solution heated 3 times to 35°C by microwave irradiation.
Figure 4.9B The asynchronous map generated from a VT-FTIR run (25-55°C)
75
of a p-lg solution heated 3 times to 35°C by microwave irradiation.
Figure 4.10A Overlaid deconvolved infrared spectra o f P-lg that subjected to
77
1.3.5.7, and 10 cycles of microwave irradiation till 75°C/cycle.
Figure 4.1 OB Overlaid deconvolved infrared spectra o f P-lg that subjected to
78
1.3.5.7, and 10 cycles o f conventional heating till 75°C/cycle.
Figure 4.11 A Overlaid FTIR difference spectra o f p-lg as a function o f
78
irradiation cycles till 75°C, after subtraction o f the spectrum recorded at 30°C.
Figure 4.1 IB Overlaid FTIR difference spectra o f P-lg as a function o f
79
C.V.H. cycles till 75°C, after subtraction o f the spectrum recorded at 30°C.
Figure 4.12A The synchronous 2D IR spectra generated from the difference
80
spectra in figure 4.11 A.
Figure 4.12B The asynchronous 2D IR spectra generated from the difference
81
spectra in figure 4.11 A.
Figure 4.13A The synchronous 2D IR spectra generated from the difference
82
spectra in figure 4.1 IB.
Figure 4.13B The asynchronous 2D IR spectra generated from the difference
83
spectra in figure 4.1 IB.
Figure 4.14 Comparison between the effect o f 10 microwave irradiation and
85
conventional heating cycles till 75 °C on the extent o f aggregation o f P-lg.
Figure 5.1 Hemoglobin with the subunits displayed in ribbon representation
94
Figure 5.2 FSD spectrum o f Human hemoglobin in D 2 O at 25°C in the amide I’
97
absorption region.
Figure 5.3 FSD-FTIR spectrum o f Hb in the amide I absorption region o f Hb
98
heated twice to 54 C° by MW or C.V.H.
Figure 5.4 FSD-FTIR spectrum o f Hb in the amide I absorption region o f Hb
99
heated 4 times to 54 C° by MW or C.V.H.
Xll
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Figure 5.5 A plot o f the peak height o f the 1683 cm '1in the infrared spectra o f
100
hemoglobin heated for 4 cycles by MW or by C.V.H at 35, 48 and 54°C.
Figure 5.6 A plot o f the peak height o f the 1653 cm '1in the infrared spectra o f
100
hemoglobin heated for 4 cycles by MW or by C.V.H at 35, 48 and 54°C.
Figure 5.7 Fluorescence emission spectrum o f native hemoglobin at 25 °C,
101
excited at 280 nm.
Figure 5.8 Fluorescence spectra o f hemoglobin at 48°C using an excitation
103
at 280 nm after 4 cycles o f MW or C.V.H.
Figure 5.9 Fluorescence spectra o f hemoglobin at 54°C using an excitation
103
at 280 nm after 4 cycles o f MW or C.V.H.
Figure 5.10 Far UV CD spectrum hemoglobin at 25°C.
105
Figure 5.11. Far UV CD spectrum hemoglobin at 54°C after four heating
106
cycles, by MW or by C.V.H.
xiii
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LIST OF ABBREVIATIONS
2D
Two-dimensional
2D IR
Two-dimensional infrared
P-lg
P-lactoglobulin
CaF2
Calcium fluoride
CD
Circular dichroism
C.V.H
Conventional heating
D20
Deuterium oxide
DTGS
Deuterated triglycine sulfate
FSD
Fourier self-deconvolution
FTIR
Fourier transform infrared
Hb
Hemoglobin
IR
Infrared
MW
Microwave
Tm
Transition Temperature
UV
Ultra-violet
VT-FTIR
Variable-temperature Fourier transform infrared spectroscopy
AG
Free energy change
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CHAPTER 1
GENERAL INTRODUCTION
Ever since Underwood and Prescott instituted thermal processing o f foods in
1885, the food processing industry has examined different means o f its implementation.
The development o f freezing as a large-scale commercial entity took place in 1930s. The
later 1940s and 1950s were the years o f ionizing radiation. Processes such as hydrostatic
cooking and removing water from foods at low cost were expanded during 1960s
(Decareau, 1985).
Microwave heating was relegated to restaurant kitchens and to the laboratory uses
since its commercial development 1946 and it was not until the early 1960s when a useful
continuous radio frequency applicator system devised that its use gained popularity.
Microwave irradiation is a unique source o f energy because it produces heat that
penetrates deeply into materials being processed. This property results in significant
reduction in thermal processing times often by as much as an order magnitude compared
to conventional heating methods (Decareau and Peterson, 1986).
Microwave processes have gradually achieved recognition by the food industry
for blanching, cooking, drying, pasteurizing, sterilizing, and thawing o f bulk food
products (Decareau, 1985) as it offers a considerable potential for swift and uniform
heating o f food products, reduced thermal inactivation o f nutrients, increased retention of
food quality factors and is more energy competent compared to conventional thermal
processes (Mudgett, 1986). Other advantages o f microwave and radio frequency heating
systems are that they can be turned on or off instantly, and the product can be pasteurized
after being packaged.
Industrial microwave processing of food did not develop rapidly due to lake o f
information on microbial safety and product quality. Furthermore, to this day there is a
^
controversy whether microwave inactivation o f microorganisms occurs via thermally
induced effects o f microwave treatment or whether a nonthermal effect o f microwave
heating exists. Although researches rebut any molecular effects o f electric fields
^
compared with thermal energy using classical axioms of physics and chemistry, numerous
1
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theories have explained how electromagnetic fields might kill microorganisms without
heat as summarized in a review by Knorr et al., 1994.
In latest decades, many studies have focused on different procedures, approaches,
techniques, biological systems and experimental designs to distinguish thermal and
nonthermal effects o f microwave heating to resolve this controversial question. This work
focuses on the use o f two protein-based model systems, P-lactoglobulin and hemoglobin,
to delineate the existence and mechanism o f nonthermal effects on protein unfolding
employing FTIR, circular dichroism (CD), and fluorescence spectroscopy to track
changes in protein conformation as a function o f microwave treatment. Two dimensional
correlation analyses will also be employed to establish the sequence o f event leading to
protein unfolding as a function o f conventional and microwave heating treatments.
Therefore, this thesis is structured in four main sections. The first is a review o f literature
concerning the microwave effects and proposed mechanisms o f these effects. The
subsequent chapters consist o f three papers which will be submitted for publication.
Chapter 3 addresses the existence o f a nonthermal effect o f microwave irradiation on
globular proteins (p-lactoglobulin) by comparing the effects microwave treatment to the
conventional heating on P-lactoglobulin in solution. Chapter 4 describes the sequence o f
events leading to p-lactoglobulin unfolding and aggregation using two-dimensional
correlation spectroscopy.
Chapter 5 examines the effect microwave irradiation on the
secondary and tertiary structure o f hemoglobin using FTIR, CD and Fluorescence
spectroscopic techniques. In the final chapter, the conclusions drawn from the results
obtained in chapters 3-5 will be exploited to delineate the mechanism o f nonthermal
effects o f the microwave irradiation on the proteins investigated.
2
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CHAPTER 2
LITERATURE REVIEW
2.1 Electromagnetic Radiation
Electromagnetic radiation is made up o f two wave motions perpendicular to each
other. One is a magnetic (M) wave; the other is an electric (E) wave. Electromagnetic
waves are generated by an alternating or cyclic movement o f electrons which causes
changes in the electric and magnetic fields and results in the propagation of
electromagnetic radiation. The energies associated with E and M are equal, but most
optical effects are concerned with the electric waves. Table 2.1 summarizes some o f the
properties o f electromagnetic radiation.
2.2 Microwave
Microwave is the name given to electromagnetic waves arising as radiation from
an electrical distribution across a broad spectrum o f frequencies ranging between 300
MHz and 300 GHz (Figure 2.1).
Millimeter
waves
Radio frequencies
Microwave
-M — M -
10s
Visible light
Infrared
Ultraviolet
-> i
r* --------------- M
X-rays
---------------------
106 107 108 109 1010 1011 I012 1013 1014 1015 1016 1017
Frequency (Hz)
Figure 2.1 Electromagnetic spectrum.
3
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Table 2.1 Properties o f electromagnetic radiation
Name
Dispersed
Generated by
Particular
Detected by
by
(Electrons)
(Neutrons)
Properties /Uses
Acceleration o f thermally
Electric
Photography;
Deflected by electric
produced electrons by a
and
fluorescence
and magnetic fields;
high voltage
magnetic
used
fields
microscope
Nuclear reactor
Photography
(difficult);
in
electron
Scattered by nuclei;
counting
diffracted by crystals
devices
X-ray
Rapid
deceleration
fast-moving
changes
of
electrons;
in
energy
innermost
Photography;
Can
ionization chamber
matter; reflected and
of
diffracted
by
crystals; scattered by
orbital
electrons
Ultraviolet
penetrate
electrons; EXAFS
Electronic transitions of
Quartz,
Photography;
atoms and molecules
fluorite
photoelectric
Absorbed by glass
cell;
fluorescence
and
conjugated
molecules; can cause
many
chemical
reactions;
UV
spectra
Visible light
Rearrangement o f outer
Glass
orbital electrons in atoms
Eye;
photography;
photocell
action; microscopy;
and molecules
Infrared
Microwaves
Radio waves
Change
of
visible spectra
molecular
Rock salt
Photography
by
rotational and vibrational
special plate; special
energies
heating effect
Special electronic devices
Paraffin
Valve
such
wax
arranged
as
klystron tube;
circuit
as
electron spin reorientation
microwave
in a magnetic field
point-contact diodes
Oscillating
electrons
in
special circuits coupled to
radio aerials; nuclear-spin
reorientation
Can cause chemical
in
Tuned
receiver;
oscillatory
electric circuit
Radar
communication;
ESR measurements
Radio
communications;
NMR measurements
a
magnetic field
4
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2.2.1 History of Microwaves
Michael Faraday found the electromagnetic field in 1832; however it was not until
1873 that James Clark Maxwell predicted mathematically the existence and behavior o f
radiowaves. In 1940, at Birmingham University, John Randall and Harry Boot produced a
functioning prototype o f the cavity magnetron (the microwave generator). In August
1941, the earliest production model was shipped to the United States.
The discovery o f Dr. Percyl Spencer that microwaves could be efficiently applied
to the heating o f various materials if the energy were confined led to his building the
earliest microwave oven using a farmer’s milk can (Goldblith and Decareau, 1973).
Sherman (1946b), described microwave irradiation as “electronic heat”, and outlined its
potential application to the food industry. The commercial development of the microwave
oven in the mid-1940s, operating first at 2450 MHz, brought new prospects for the utility
of radio frequency heating of a variety of foods. Microwave heating applications are
limited to a discrete set o f frequencies reserved for industrial, scientific, and medical
(ISM) uses due to the overlap o f microwave frequencies with the radio frequencies used
for telecommunication. The two most common frequencies used within North America
are 915 MHz and 2450 MHz (Decareau, 1985).
2.2.2 Microwave Heating
Microwave heating is also referred to as “radio frequency” or “electronic”
heating and is related not merely to the dielectric properties o f the materials such as
foods, but to its electrical transmission properties as well (Decareaul985). Microwave
heating or dielectric heating refers to the heating that come to pass in a nonconductor due
polarization effect at frequencies between 300 MHz and 300 GHz (wavelength between
lm and 1mm respectively). An essential difference between capacitive (macrowave) and
microwave heating is related to primary to the frequencies used (1 to 300 MHz in
macrowave heating), and the comportment in which heating is carried out (in microwave
heating a closed cavity or oven is often used). In capacitive heating the material is usually
placed between electrodes and irradiated with frequencies between 300 MHz and 300
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
GHz) (Decareau, 1985). Microwaves in themselves are not heat; it is the material that
absorbs microwaves converts the energy to heat. In food system, the heat is generated by
interface between microwaves and polar molecules or ions.
Abstractly, microwave heating o f foods uses two basic phenomena, first, coupling
the energy by the product with the electromagnetic field, second attenuation o f absorption
of the coupled energy within the product. Water is the ubiquitous polar molecule in the
majority o f foods. Since water molecules have negative and positive ends, in the presence
of a microwave electric field, these charges attempt to line up with the electric field. As
the microwave field reverses it’s polarity millions o f times per second, the water
molecule, constrained by the natural structure o f the food o f which it is a part of, begins
doing a flip-flop movement millions times per second. In doing so, week hydrogen bonds
are disrupted and heat is generated by the molecular friction and is dissipated rapidly
through out the food materials. Because the molecules are forced to rotate first, there is a
slight delay between the absorption o f microwave energy and the development o f linear
momentum, or heat.
Other constituents such as salts also contribute to heat generation by either friction
or high speed electrophoretic migration in the electric field. Charged ions are influenced
by microwave electric field, so they migrate first in one direction then in the contrary
direction as the electric field is reversed (Figure 2.2).
There are some unimportant secondary effects o f microwaves, including ionic
conduction, which are negligible in producing heat.
6
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A lternatin g
:lectric
fie ld
IT
0--\
Ionic In teraction
R otation
D ip olar In teraction
Figure 2.2 Proposed mechanism o f microwave heating (source: Buffler, 1993).
2.3 Dielectric Properties of Food Materials and Electric Field Interactions
The fundamental dielectric properties o f food are correlated to their chemical
composition, and modified to some degree by its physical structure, and are very sensitive
to the irradiation frequency and temperature o f the food. The dielectric constant o f foods
is primarily determined by free water and salt contents and other electrical properties that
affect energy coupling from an electromagnetic field within the product. Other electrical
properties of essential concern in microwave heating are the product intrinsic impedance
relative to free space, which affects the level o f power transmission and reflection at
product surfaces, and attenuation factor, which determines the level o f power absorption
within the product as a function of depth from the surface (Decareau, 1985).
The dielectric properties o f common concern are the dielectric constant E (a measure of
the ability o f the material to store microwave energy) and the dielectric loss factor E (a
measure o f the ability o f the material to dissipate this energy as heat), which the real and
imaginary parts of the relative complex permittivity Er given by
Er = E - z'E
The dielectric properties o f materials dictates to a great extent the behavior o f the
materials subjected to radio frequency (RF) or microwave fields for purposes o f drying or
7
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heating the material (Nelson and Datta, 2001).
The dielectric properties o f foods fluctuate extensively with, temperature,
moisture content, frequency and physical status (Table 2), and are dependent on the
chemical composition and particularly on the presence o f mobile/ions and the permanent
dipole moments linked with water and any other molecules making up the material o f
interest. In hygroscopic material such as foods, the amount o f water in the material is by
and large a governing factor.
Table 2.2 Microwave dielectric properties o f water at indicated temperatures
Frequency
E at 20°C
E at 20°C
E at 50°C
E at 50°C
0.6
80.3
2.75
69.9
1.25
1.7
79.2
7.9
69.7
3.6
3.0
77.4
13.0
68.4
5.8
4.6
74.0
18.8
68.5
9.4
7.7
67.4
28.2
67.2
14.5
9.1
63.0
31.5
65.5
16.5
12.5
53.6
35.5
61.5
21.4
17.4
42.0
37.1
56.3
27.2
26.8
26.5
33.9
44.2
32.0
36.4
17.6
28.8
34.3
32.6
(GHz)
The dielectric loss o f aqueous solutions as a function o f microwave frequencies is
attributable to the effects o f dipole rotation and ionic charge migration, with ionic
conduction having a superior influence at lower frequencies and ionic conduction losses
declining with increasing frequency. The dipole loss also decreases as the temperature
rises.
Bound water contributes less to the dielectric constant o f free water in the
microwave region, because the bound water is not capable o f responding to the
alternating electric field at such frequencies. For fairly low-moisture foods, the dielectric
constant and loss factor increases with increasing moisture content. For high-moisture
8
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foods, in which bound water plays an inconsequential role, the properties are influenced
not just by the water content but also by dissolved constituents and ionic conduction,
although the contribution o f charge carriers to the dielectric loss is very little at
frequencies above 2 GHz (Nelson and Datta, 2001).
2.4 Microwave Effects
According to the literature, two types o f effects can be ascribed to microwaves,
and referred to as thermal and nonthermal effects (Frohlich, H. 1980, Cleary, S.F 1989,
Marani, E. and Feirabend, H.K.P. 1994, kirschvink, J.L. 1996). As previously discussed,
the microwaves have the ability to penetrate the food and generate heat by friction
resulting from the oscillation o f water dipole, which will try to align with the field
resulting in frication with other food constitutes; this is the macroscopic therm al effect of
increasing temperature within the material. N ontherm al effects under the application of
electromagnetic radiation refer to observable changes that do not involve a significant rise
in temperature as in the case o f ionizing radiation. According to Tong, microwave
nonthermal effects are defined as the existence o f additional destruction effects that
cannot be explained by heat alone (Tong, 1996).
The microwaves that we use to heat food have wavelengths that are about 1
hundredth o f a meter long and have frequencies of about 2.5 billion Hertz. Higher
frequency ultraviolet radiation begins to have enough energy to break chemical bonds. Xray and gamma ray radiation, which are at the upper end o f magnetic radiation have very
high frequency in the range o f 100 billion billion Hertz and very short wavelengths 1
million millionth o f a meter. As the wavelength increases and frequency decreases, not
enough energy is accessible to break chemical bonds. Roughly, one electron volt of
energy is required to break a covalent bond to produce an ion pair. Ultraviolet, visible,
and perhaps infrared rays have energy to break weak hydrogen bonds. But microwaves
radiation does not have adequate energy to break any chemical bonds and for that reason
belong to the group o f non-ionizing forms o f radiation (Anantheswaran and Ramaswamy,
2001 ).
Risman (1996) proposed that any nonthermal effect must not be explicable by
macroscopic temperatures, time-temperature histories or gradients. Goldblith and Wang
9
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showed no differences in inactivation o f E.coli and B.subtilis exposed to the same timetemperature conditions o f microwave and conventional heating.
Lechowich et al. also studied the exposure o f S.faecalis and S.cerevisiae to
microwave at 2450 M Hz and under conventional heating, and concluded that the
inactivation by microwaves could be explained solely in terms o f heat generated during
the exposures.
Vela and W u exposed various bacteria, actinomycetes, fungi, and bacteriophages
to microwave at 2450 MHz both in the presence and absence o f water, and brought to a
close that microorganisms were inactivated only in the presence o f water. This finding led
them to conclude that microorganisms were killed only by the thermal effect and most
likely there was no nonthermal effect. However, they added that if a nonthermal effect
existed, then water would be essential to potentate it, as cell constituents other than water
did not absorb satisfactory energy to kill microbial cells (Anantheswaran and
Ramaswamy, 2001).
An abundance o f studies have been conducted on the effects o f microwave heating
on food and its constituents since the middle o f the 1920s, and these have yet to resolve
the controversial issue on nonthermal microwave effects.
The existence o f biological effects arising from the nonthermal effect o f
microwave irradiation is still contentious, to a certain extent because o f a lack o f the
linear dose response relationships.
Much effort has been devoted to studies that have attempted to demonstrate the
existence of nonthermal effects o f microwave irradiation by containing end-point
temperatures below thermal death points o f microorganisms under investigation. Early
reports by Beckwith and Olsen (1931); Fabian and Graham (1933); Yen and Lui (1934);
Fleming (1944); Nyrop (1946); Carpenter (1958);Susskind and Vogelhut (1959), initiated
this hypothesis for both procaryotes and eucaryotes.
Studies o f the effects o f microwaves exposure on bacteria, viruses and DNA were
carried out in the 1960s and included research on heating, biocidal effects, dielectric
dispersion, mutagenic effects and induced sonic resonance. Some o f the early
biophysicists investigating microwave absorption claimed proof o f a microwave effect
which was discrete in its biocidal effects from the effects o f external heating (Barnes
10
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1977, Cope 1976, Furia 1986).
Culkin and Fung (1975) demonstrated that E. coli and Salmonella typhimurium
could not survive in soups cooked at 915 MHz by microwave irradiation, and found that
microbial destruction occurred at lower temperature and shorter time periods when
compared to conventional heating methods. In addition its found varied effects based
upon the intensity o f the field strength at the top and bottom o f the soup containers. This
group o f workers postulated finally that factors other than thermal effects might be
involved.
Henderson et al., 1975, studied inactivation o f horseradish peroxidase using 2450
MHz microwaves by circulating carbon tetrachloride as a coolant to control the
temperature of the sample at 25°C. These researchers reported significant enzyme
inactivation at high power and reasoned that protein denaturation perhaps due to local
heat generation within the sample (Henderson et al., 1975).
Dreyfuss et al. performed two analyses by which cultures o f S. aureus were
exposed to either microwave radiation or conventional heating, for 10, 20, 30 and 40 sec.
o
until the samples reached 46 C. The cultures were then lysed and the enzymatic activity
o f the lysates was analyzed. The production o f thermonuclease was also examined at
various levels o f exposure o f cells to microwave radiation. Dreyfuss et al. determined that
microwaves produce different effects on the cells than conventional heat treatment. This
result showed that the effects o f microwave radiation on S. aureus cannot be explained
exclusively by thermal effects (Dreyfuss, et al., 1980).
Khalil and Villota, 1988 questioned the theory o f nonthermal effects and studied
the relative effectiveness of conventional and microwave heating for inactivation
microorganisms by comparing the injury and recovery o f Stphylococcus aureus during
microwave and conventional heating and concluded that microwave-heated cells suffered
a larger injury as well as greater membrane damages.
Levina and co-workers (1989) studied microwave effects on the development of
the protozoa Spirostum sp. cell population and proposed that the irradiation affected the
population's own growth control mechanism, and that the effect depended on the stage
and other particulars o f the population development.
The inactivation o f commercial soybean lipoxygenase was studied at various
11
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temperatures
using
conventional
and
microwave heating
by
Kermasha et al.
Lipoxygenase inactivation was assessed using first-order reaction kinetics. This
experiment showed that microwave heating resulted in a more rapid inactivation o f the
enzyme. Higher enzyme inactivation rates under microwave heating conditions were
ascribed to possible nonthermal effects (Kermasha et al., 1992).
Aytekin et al. exposed suspensions of Bacillus subtilis and Escherichia coli cells
to microwave radiation at 2450 MHz for 15, 60, and 120 s with different powers 55, 165,
275, and 330 W, and observed that microwave oven caused a significant differentiation in
solutions membrane protein quantity and their electrophoretic band profiles. The
conclusion o f this research was the microwave outcome originated from thermal and
nonthermal effects o f microwave radiation (Aytekin, et al., 1994).
Kakita, 1995 reported that the microwave effect is distinguishable from external
heating by the fact that it is capable o f extensively fragmenting viral DBA, something that
heating to the same temperature did not accomplish. This experiment consisted o f
irradiating a bacteriophage PL-1 culture at 2450 MHz and comparing this with a separate
culture heated to the same temperature. The energy level o f a microwave photon is only
10' 5 eV, whereas the energy necessary to break a covalent bond is 10 eV, or a million
times higher. Based on this, it has been stated in the literature that microwave radiation is
incapable o f breaking covalent bonds o f DNA (Jeng, 1987, Fujikawa, 1992), but this has
apparently occurred in the Kakita experiment, even though this may be only an indirect
effect of the microwave irradiation. There is further evidence that point to an alternate
mechanism for causing covalent bond breakage in DNA without invoking the energy
levels o f ionizing radiation (Ishibashi 1982, Watanabe 1985, 1989, Kakita 1995, Kashige,
1990 1994, 1995). Still, no theory currently exists to explain the phenomenon o f DNA
fragmentation by microwaves.
In a comparative study o f the inactivation o f L.Plantarum using microwave and
conventional heating at 50°C for 30 min, Shin and Pyun, 1997, found considerable
nonthermal effects under pulsed microwave heating conditions
A microwave heating system was evaluated for subjecting liquid foods to
continuous-flow heat-hold-cool pasteurization process by Tajchakavit et al., 1997.
Results indicated that the system could be used to study the inactivation kinetics under the
12
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microwave heating conditions as well as under isothermal hold, and that the D-values at
60 C o f microwave heating were considerably shorter than those obtained under
conventional heating conditions. Microwave inactivation o f enzyme and destruction of
microorganisms in apple juice during continuous-flow microwave heating conditions
were found to be much faster than in conventional thermal mode at any given
temperature. These results suggested some contributory nonthermal effects to be
associated with microwave heating. (Tajchakavit et al., 1997).
The effect o f microwave irradiation at 10.4 GHz on a thermostable enzyme (figalactosidase) was experimentally tested by La Cara et al., 1999 to study the effects o f
microwaves on protein stability. They observed that microwave irradiation induced an
irreversible inactivation in the enzyme at 70°C compared to conventional heating at the
same temperature. This inactivation was dependent on the intensity o f the microwave and
as well as the protein concentration. Inactivation was not observed following
conventional heating at the same temperature (La Cara et al., 1999).
Additional evidence suggests that cells processes can be influenced by weak
electromagnetic fields (EMFs). EMFs appear to represent a global interference or stress to
which a cell can adapt without catastrophic consequences. The age and state o f the cell
can profoundly affect the EMF bioresponse (Goodman et al., 1999).
Koutchma et al. in comparative experimental evaluation o f microbial destruction
in continuous-flow microwave and conventional heating systems showed that under a
variety o f experimental conditions, the surviving microbial population o f Escherichia coli
following the heat treatment was enumerated. The obliteration data were then combined
with experimentally measured time-temperature data to compute the associated decimal
reduction times. D-Values for continuous-flow microwave heating were considerably
lower in comparison with both continuous flow steam heating and batch heating. Hence,
there was evidence that microbial lethality under microwave heating condition can not be
fully accommodated by conventional models employing thermal kinetics data (Koutchma
et al,
2 0 0 1
).
When microwave irradiation effect were compared to the conventional heating
effect on Escherichia coli at different temperature, no cell death was observed at 35 °C,
whereas at 45, 47 and 50 °C, the death rates o f Escherichia coli exposed to microwave
13
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irradiation were higher than those obtained in conventional heat sterilization at the same
temperature. Even though the author could not stipulate the exact mechanism underlying
the nonthermal effect o f microwaves, he proposed that the microwaves either caused ions
to accelerate and collide with other molecules or caused dipoles to rotate and line up
rapidly w ith alternating (2450 million times/s) electric field resulting in a change in
secondary and tertiary structure o f proteins o f microorganisms (Sato, 2001).
Olsen et al. compared conventional and microwave heat treatment o f Aspergillus
niger, Rhizopus nigricans, and Penicilium sp. and found that to accomplish a given level
of killing, lower temperatures were achievable in the microwave treatment than with
conventional heat treatment (Anantheswaran and Ramaswamy, 2001).
. Berzhanskaya and co-workers found a suppression o f bioluminescence o f
Photobacterium leiognathi at 36.2-55.9 GHz microwave irradiation. Results o f the
studies on Enterobacter aerogenes and E. coli B showed that microwave radiation
inhibited or stimulated protein, DNA and RNA synthesis and cell growth (Pakhomov et
al.,
2 0 0 1
).
Inactivation kinetics of an a-amylase enzymatic time-temperature integrator (TTI)
from Bacillus subtilis (BAA) under continuous-flow microwave and conventional hold
conditions were evaluated and compared by Tong et al. Comparing the D-values between
continuous-flow microwave and thermal holding, enzyme destruction occurred much
faster under continuous-flow microwave heating condition than under conventional
thermal heating condition. Hence, there is evidence that microbial lethality under
microwave heating conditions cannot be fully accommodated by conventional models
employing thermal kinetics data. The results indicated further that the possibility o f exist
of nonthermal or enhance thermal microwave effects (Tong et al., 2002).
The nonthermal effects o f microwave irradiation on enzyme-catalyzed reactions
have been evaluated by keeping the reaction temperature constant during irradiation,
microwave irradiation was found to increase the initial reaction rates by 2.1-4.7 times at
all hydration levels. It is also shown that microwave irradiation can be used in
conjunction with other strategies like pH tuning and slat activation for enhancing initial
reaction rates (Ipsta et al., 2003).
14
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2.5 Mechanisms o f Inactivation/destruction o f Microorganisms by Microwave
irradiation
The energy absorption from microwave and radio frequency can raise the
temperature o f the food high enough to inactivate microorganisms for effective
pasteurization or sterilization. The mechanisms contributing to microbial death or enzyme
inactivation can be attributed to three major effects:
2.5.1 Thermal Effects
An increase in temperature can result in changes in stability and functionality of
macromolecules and consequently the biological processes in the cells/molecules.
Increasing temperature can lead to protein and nucleic acids denaturation resulting in
inactivation/destruction of the microorganisms.
2.5.2 Kinetic Effects
The net charges o f cells may be influenced by the electromagnetic field o f
microwaves and might lead to a quick oscillation that exceeds the elastic limitation o f the
cell wall resulting in the disruption o f the cell membrane (Carroll and Lopez, 1969; Khalil
and Villota, 1988; Palaniappan et al., 1990). This may lead to modification o f membrane
permeability, leakage o f cellular contents, loss o f cell functionality and, ultimately, death
of cells. The passage o f electron current could also induce the alteration o f growth and
nerve processes o f cells (Cope, 1976). In addition, a re-alignment o f lipids in the field
may cause the breakdown o f lipid orientation (Wildervanck et al., 1959; Teixeira-Pinto et
al., 1960; Olsen et al., 1966; Rosen, 1972).
2.5.3 Chemical Effects
The quantum energy levels of microwaves are several magnitudes lower than that
required to break chemical bonds, hence it is unlikely that microwaves could break any
type o f chemical bond in foods (Rosen, 1972; Pomeranz and Meloan, 1987). However, it
is possible that free radicals o f oxygen, hydrogen, hydroxyl and hydroperoxyl may be
formed by simultaneous absorption o f energy (Olsen et al., 1966; Rosen, 1972).
Microbial cells may also be selectively heated by microwaves depending on their
chemical composition and the surrounding medium (Carroll and Lopez, 1969).
In view o f food as a whole, it is feasible that microwave energy may be
concentrated in micro-or macroscopic layers o f food resulting in unconventional chemical
15
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reactions due to higher local temperatures than the average temperature (Lorenz, 1976).
Kozempel et al. suggested four predominant theories to explain nonthermal
inactivation by microwaves: selective heating, electroporation, cell membrane rupture,
and magnetic
field
coupling. The
selective heating theory
affirms that solid
microorganisms are heated more efficiently by microwave than the surrounding medium
and are accordingly killed more readily. Electroporation is caused when pores form in the
membrane o f the microorganisms due to electrical potential across the membrane,
resulting in leakage. Cell membrane rupture is associated with the voltage drop across the
membrane causing it to rupture. In the magnetic field coupling theory, cell lysis occurs
due to coupling o f electromagnetic energy with critical molecules within the cells, leading
to disruption o f the internal components o f the cell.
2.6 Microwave Effects on Protein Structure
Tertiary structure refers to the complete three-dimensional structure of the
polypeptide units o f a given protein. Included in this description is the spatial relationship
of different secondary structures to one another within a polypeptide chain and how these
secondary structures themselves fold into the three-dimensional form o f the protein.
Secondary structures o f proteins often constitute distinct domains. Therefore, tertiary
structure also describes the relationship o f different domains to one another within a
protein. The interactions o f different domains are governed by several forces: These
include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van
der Waals forces. Although the mechanism o f the nonthermal effect is unknown, violent
motion o f dipoles in molecules by microwave field seems to destroy structures of
polyatomic molecules such as protein and phospholipids. Microwaves either cause ions to
accelerate and collide with other molecules or cause dipoles to try to rotate and line up
with rapidly alternating electrical field resulting in 2,450 million times oscillations per
second (Sato, et al., 1996).
Exposure o f two thermophilic and thermostable enzymes, to microwave
irradiation resulted in a nonthermal, unalterable and time-dependent inactivation o f both
enzymes below their optimal operating temperatures. The inactivation rate was correlated
to the energy absorbed and is sensitive to the enzyme concentration, suggesting that
16
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microwaves stimulate protein structural rearrangements that are not related to temperature
(Porcelli et al., 1997).
Akin notions can be applied for determining kinetic parameters during microwave
heating; nevertheless, non-isothermal heating conditions are involved in this case. The
food constituent principally experiences changing temperature with time. The procedure
is more complex than with the isothermal procedure. There have been only a small
number of studies o f kinetics during microwave heating.
Bohr reported that at low temperatures, the re-folding o f cold denatured (3-lg is
enhanced by microwave treatment, while at elevated temperatures the denaturation o f Plg from its folded state is enhanced by microwave treatment. In the second case, a
negative temperature gradient is needed for the denaturation process, suggesting that the
effects o f the microwave are nonthermal (Bohr et a l, 2000). This supports the concept
that coherent topological excitations can be real in proteins. Also there is experimental
evidence that microwave irradiation could speed up rates o f folding and unfolding o f
other globular proteins in solutions (Bohr et al., 2000).
Exposure to microwave radiation increases the aggregation of bovine serum
albumin in vitro in a time-and temperature-dependent manner (David et al., 2003). The
same group also claimed that microwave irradiation can induce amyloid fibril formation
in vitro, at least under the non-physiological conditions used in the experiment (David et
al., 2003).
2.7 Mechanisms of Nonthermal Effects
Very little is known about the molecular mechanisms involved in the presumed
nonthermal effects which might engage direct energy transfer from the electromagnetic
field to the vibrational modes o f macromolecules altering their conformation (Taylor,
1981).
It is
conceivable
that
direct
energy
absorption
could
occur
from
an
electromagnetic field into a vibrational mode of a protein molecule. For small molecules
with vibrational modes in the THz range it is exceptional for the lifetime o f vibrational
excited state to exceed 1000 oscillations (Chronister & Crowell, 1991). The rate of
migration o f energy is dependent upon the anharmonic terms and these are likely to be
17
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very large for macromolecule vibrations.
Many nonthermal effects following exposure of biosystems to microwave have
been reported, among them changes o f the activity of Ca2+ dependent K+ channels
(Fesenko, 1995), alterations o f membrane structure and function (Phelan et al., 1994,
Persson et al., 1992), permeability modification o f liposomes (Saalman, et al., 1991,
Orlando and Mossa 1994), and isolated cells (Phelan, et al., 1992) have been described.
It is well accepted that exposure to the electromagnetic fields in the microwave region
imposes stresses on the living cells when the specific absorption rate (SAR) is beyond a
level sufficient to cause the temperature o f the cells and tissues to be significantly
elevated.
The polypeptide chains o f a protein follow topological constraints which enable
long range excitations in the form o f wring modes o f the protein backbone. Wring modes
of proteins o f specific lengths can therefore resonate with molecular modes. It has been
suggested that protein folding is governed by a fast hydrophobic collapse followed by a
slower annealing (Dill et al., 1995). Bohr suggested that the initiation o f protein folding is
a resonance phenomenon, and that protein folding takes place when the amplitude o f the
resonance mode exceeds a certain threshold. Folded structures become stabilized by van
der Waals interactions, hydrogen bonding, salt bridges and disulfide bonds and even in
some cases by dynamic forces (Bohr, 1997). Wring modes will divide a protein chain into
regions, which are subject to large and small torsional amplitudes. An amino acid
substitution in a region with high torsional amplitude is more likely to have a modifying
effect on the folding o f protein than a substitution in a region o f low torsional amplitudes.
A phase transformation in proteins can be initiated by long range collective wring modes
o f the backbone. Bohr suggested that this is an important part o f the underlying
mechanism behind the transformation o f a protein from the unfolded to the folded
structure. He also assumed that a wring mode is being pumped to levels o f higher and
higher amplitude, and that eventually this wring mode becomes unstable in favor o f
curvature. The nature o f such a transition is better characterized as being catastrophic than
entropic. Therefore, the primary reason for the transition is not a change in entropy,
rather, a resonator is responsible for pumping the twist mode to a higher and higher level.
Folded proteins can also maintain wring modes, and that it is the disappearances o f these
18
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wring modes upon lowering the temperature that lead to cold denaturation o f certain
proteins. Only contributions to the change in free energy, when going from an unfolded to
a folded, and from folded to an unfolded protein, is caused by their differences in wring
activity. Proteins can sustain eigenmodes involving a wringing and the eigenfrequency of
the wring mode may be as high as values typical for excitation o f molecular structures.
This points to an intriguing possibility that the transition from the unfolded to the folded
state o f a protein, can occur when a wring mode o f the protein backbone becomes
unstable to curvature (Bohr, 1997).
Large amount o f structural motion in proteins at physiological temperatures has
been demonstrated by X-ray and nuclear magnetic resonance experiments; therefore, a
possible mechanism for nonthermal microwave-biological effects is that the process o f
microwave absorption transfers energy from the electromagnetic field to the vibrational
modes o f macromolecules, altering mode structure and possibly macromolecular
conformation. Such changes can be a result o f a particular vibrational mode being
selectively excited in a nonthermal energy distribution resulting in an increase in the
protein distortion yielding a new conformation.
Surrounding water molecules exposed to hydrophobic regions on protein
molecules adopt hydrogen-bonded pentagonal structures. An increase in the temperature
o f the water can have significant consequences for protein conformation. The breaking of
hydrogen bonds within the water pentagonal structure may cause it to give way to the
bulk water state (Chattopadhyay et al., 1997). As a result, hydrophobic groups on the
surface o f the protein may be able to interact with each other to a greater extent and the
protein may adopt a different conformation.
The mechanism o f biological effects o f extremely-low-frequency electric and
magnetic fields may involve induced changes o f Ca2+ transport through plasma
membrane ion channels. Kim et al., 1998 studied the effects o f externally applied, lowintensity 60 Hz electric (E) fields (0.5 V/m, current density 0.8 A/m2) on the agonistinduced Ca
fluxes o f HL-60 leukemia cells. Their results demonstrated that low-
intensity electric fields can alter calcium distribution in cells, m ost probably due to the
effect on receptor-operated Ca2+ and/or ion channels (Kim et al., 1998).
Daniells et al., 1998, showed that using a pulsed microwave exposure at 750 MHz
19
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o f cells resulted in the activation o f heat shock genes, and lower power levels tended, in
general, to induce larger responses, which is contrary to the results expected if the effect
were simply due to heating. They concluded that microwave radiation causes the
activation o f cellular stress responses, as a result o f increased levels o f protein structural
alteration by mechanisms other than heating. The microwave pulses can cause unfolding
to occur only through transient heating o f both the molecule itself and its aqueous
environment. This can occur either through the resonant absorption o f energy initially in
the peptide chain or through resonant absorption o f the aqueous environment.
Microwaves are likely to cause “hotspots” in particular parts o f a cell, such as the
membrane (Liu and Cleary 1995). Such localized absorption could result in transient
temperature rises which would not be measurable, but would be sufficient to induce
protein unfolding (Laurence et al., 2000).
(Laurence, et al., 2000).
De Pomerai et al. showed that prolonged exposure to low-intensity microwave
fields can induce heat-shock responses in the soil nematode Caenorhabditis elegans.
Therefore, they suggested instead that the induction o f heat-shock proteins (HSPs)
described here could involve nonthermal mechanisms. These could include microwave
disruption o f the weak bonds that maintain the active folded forms o f proteins; enhanced
production o f reactive oxygen species (known to be inducers o f HSPs; or interference
with cell-signaling pathways that affect HSP induction (by heat-shock-factor activation).
All these mechanisms are testable using the functional genomic tools that are available in
C. elegans. Because o f the universality o f the heat-shock response, a similar nonthermal
induction might also occur in human tissues exposed to microwaves, a possibility that
needs investigation (De Pomerai et al., 2000).
A group o f researchers, in experiments using continuous and pulsed microwave
sources have demonstrated that nonthermal microwave exposure activates the heat shock
response. Activation o f the heat shock response is a strong indicator that microwave
sources cause partial unfolding o f proteins within cells. Steel, et al., in exploring the
effects of stress on biological systems, demonstrated that stress will affect the
conformation o f biological molecules such as proteins, which will lead to an effect on
their function (Steel et al., 2002)
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2.8 Effect o f Physiochemical Conditions on Microwave Treatment
Procelli et al., 1997 in their experiment on thermophilic and thermostable
enzymes to discriminate between thermal and nonthermal microwave effects showed that
the effect o f the electromagnetic field does not depend on the enzymatic concentration o f
the sample. In the same experiment, the effect o f salts on the enzyme inactivation also has
been studied by subjecting AdoHcy hydrolase and MTA phosphorylase to 10.4 GHz
microwave irradiation at 90c in the present o f 250 mM KC1 or 250 m M KH 2 PO 4 . The
two thermophilic enzymes show different behavior; the addition o f KC1 or KH 2 PO 4 to the
enzymatic solution exposed to microwave does not cause any further effect on AdoHcy
hydrolase inactivation. On the other hand, KH 2 PO 4 exerts a moderate protection towards
microwave inactivation o f MTA phosphorylase while KC1 enhances the inactivation
process. Furthermore, a similar experiment, performed with 250 mM NaCl and 250 mM
Na 2 S 0 4 revealed that after 1 hour o f microwave irradiation o f MTA phosphorylase at
90°C, Na 2 S 0 4 exerts a moderate protection (76% residual activity), while NaCl causes an
increase o f the inactivation o f the enzyme (34% residual activity). The protection against
microwave inactivation exerted by phosphate or its analog sulfate could be ascribed to
their role as substrate o f MTA phosphorylase.
In synopsis, it seems that there are rationales to deem that the microwave effect
does indeed exist, even if it cannot yet be adequately explained.
2.9 Microwave and Human Health
Microwaves are a form of radiant energy. Although microwaves do not carry
enough energy to be ionising, they can be dangerous. However, no one really knows for
sure what the safe levels o f exposure are. A number o f laboratories in the United States o f
America have found that low level exposure to microwaves can cause cumulative effects
to the eyes, resulting in cataracts
The effects o f microwave exposure on apoptosis at nonthermal level (48h, 2450
MHz, 5 mW/cm2) have been studied by Peinnequin, et al. The results they obtained
showed a nonthermal microwave effect on Fas-induced apoptosis in human Jurkat t-cell
line, but neither on butyrate- nor on ceramide-induced apoptosis in human jurkat t-cell
line. These data show that microwave interacts either with Fas pathway between receptor
21
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and caspase-3 activation or on membrane proteins (Peinnequin, et al., 2000).
Banik et al. foimd in their review that microwave effects were established at all
biological levels, from microbial cells to animals as well as the human system. Also they
found that microwave could nonthermally induce different physiological effects and
microwaves act as promoting agents in inducing genetic changes in biosystem (Banik et
a l,
2 0 0 2
).
The human body is filled with charged biomolecules and water in nerve endings,
cell nuclei, muscles etc. which can be influenced by an electric field or magnetic field,
because, of their irregular charge distribution. Therefore, electromagnetic fields can
physically move, reorient, even alter, molecules or ions or their distribution in the body.
They can affect the rate o f chemical reactions, transport across membranes and signal
transduction. Furthermore, if charge acceleration occurs, for example, by very fast radar
pulses, the tissue itself may reradiate or scatter this energy inside the human body, thus
complicating and intensifying the radiation effects. It has been reported that ultrashort
electromagnetic pulses may cause mechanical damage to tissues through the so-called
precursor radiation, which describes the secondary bursts o f radiation that occur within
living tissue, when it is hit by e.g. radar pulses (University o f Aarhus, Denmark).
A novel study by Leszczynski, et al. about the nonthermal activation o f the
hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells
was performed. They examined whether nonthermal exposures o f cultures o f the human
endothelial cell line EA.hy926 to 900 MHz GSM mobile phone microwave radiation
could activate a stress response. Results obtained demonstrated that 1-hr nonthermal
exposure of EA.hy926 cells changed the phosphorylation status o f numerous, yet largely
unidentified, proteins. This group o f workers suggested that mobile phone radiationinduced activation o f hsp27 might facilitate the development o f brain cancer by inhibiting
the cytochrome c/caspas-3 apoptotic pathway and cause an increase in blood-brain barrier
permeability through stabilization o f endothelial cell stress fibers. They postulated that
these events, when occurring repeatedly over a long period, might become a health hazard
because of the possible accumulation o f brain tissue damage. In addition, other brain
damaging factors may co-participate in mobile phone radiation-induced effects
(Leszczynski et al., 2002).
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The effects of long term exposure to low levels o f microwaves, and their
implication to human health, will become clear only after large numbers o f people who
are being exposed to microwaves are studied for many years. Because no one can say
with certainty what levels o f exposure are safe, the course o f wisdom is to avoid exposure
to any unnecessary microwave radiation.
23
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REFERENCES
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Human Hemoglobin Studied by Fourier Transform Infrared Spectroscopy. The
journal o f biological chemistry 255, 3892-3897.
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technology for food applications. Edited by ISBN: 0-8247-0490-8 Page: 1-200.
3- Bohr, H. and Bohr, J., 2000. Microwave-enhanced Folding and Denaturation of
Globular proteins. The American Physical Society, V 61, number 4.
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studies o f a Protein. Bioelectromagnetics 21:68-72.
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D., Sewell, P., Tattersall, J., Jones, D., Candido, P., 2000. Cell biology: Nonthermal
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I.R., Jones, D., Peter, E., Candido, M., 2003. Microwave Radiation Can Alter
Protein Conformation without Bulk Heating. FEBS Letters 543, 93-97 Published by
Elsevier Science B. V. May 2003.
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effects o f EMF on cellular signal transduction-Microwave ‘Danger’to preganant
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- Dreyfuss, M. S. and Chipley, J.R., 1980. Comparison o f Effects o f Sublethal
Microwave Radiation and Conventional Heating on the Metabolic Activity of
Staphylococcus aureus. Appl. Microb. 39(1): 13-16.
13-Englander, J.J., Mar, C.D., Li, W., Englander, S.W., Kim, J.S., Stranz, D.D.,
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14-Goodman E.M.; Greenebaum B.; Marron M.T., 1995. Effects o f electromagnetic
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279-338.
15-Ipsita, R.; and Munishwar, N.G., 2003. Nonthermal Effects o f Microwaves on
protease-catalyzed Esterification and Transesterification. Tetrahedron 59, 54315436.
16-Kaufmann, B. and Christen, P., 2002. Recent Extraction Techniques for Natural
Products: Microwave-assisted Extraction and Pressurized Solvent Extraction.
Phytochemical Analysis. V.13, 105-113.
9- Kermasha, S., Bisakowski, B., Ramaswamy, H. and van de Voort, F.R., 1993.
Thermal and Microwave Inactivation o f Soybean Lipoxygenase. LebensmittelWissenschaft & Technologie, V 26, pp. 215-219.
10-Kinetics o f Microbial Inactivation for Alternative Food Processing Technologies,
Microwave and Radio Frequency Processing. U.S Food and Drug Administration,
Center for Food Safety and Applied Nutrition. June 2,2002.
11-Kim Y V; Conover D L; Lotz W G; Cleary S F, 1998. Electric Field-induced
Changes in Agonist-stimulated Calcium Fluxes o f Human HL-60 leukemia cells.
Bioelectromagnetics. 19(6), 366-76.
12-Koutchma, T., LeBail, A. and Ramaswamy, H.S, 2001. Comparative Experimental
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o f Microbial
Destruction
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Continuous-flow
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Conventional Heating Systems. Canadian Biosystems Engineering 43, 3.1-3. 8 .
17-La Cara, F., Scarfi, M.R., D ’Auria, S., Massa, R., D ’Ambrosio, G., Franceschetti,
G., Rossi, M. and De Rosa, M., 1999. Different effects o f microwave energy and
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conventional heat on the activity o f a thermophilic [3-galactosidase from Bacillus
acidocaldarius. Bioelectromagnetics 20:172-176.
18-Laurence, J.A.; French, P.W.; Lindner, R.A. and McKenzie, D.R., 2000, Biological
Effects o f Electromagnetic Fields-Mechanisms for the Effects o f Pulsed Microwave
Radiation on Protein conformation. J.Theor. Biol. 206, 291-298.
19-Leszczynski, D., Joenvaara, S., Reivinen, J., Kuokka, R., 2002. Nonthermal
activation o f the hsp27/p38MAPK Stress Pathway by Mobile Phone Radiation in
Human endothelial cells. STUK-Radiation and Nuclear safety Authority, Helsinki,
Finland. Differentiation, 70 (2-3), 120-129.
20- Li, R., Nagai, Y. and Nagai, M. 2000. Changes o f Tyrosine and Tryptophan
Residues in Human Hemoglobin by Oxygen Binding: near-and far UV circular
dichroism o f isolated chains and recombined hemoglobin. Journal o f inorganic
biochemistry 82,93-101.
21-M ohsenin, N., 1984. Electromagnetic radiation properties o f Foods and Agricultural
Products Publisher: New York Gordon and Breach, ISBN: 0677061900.
22-M udgett, R.E., 1986. Microwave Properties and Heating Characteristics o f Foods
Food Technol. 40, 84-93.
23- Peinnequin, A., Piriou, A., Mathieu, J., Dabouis, V., Sebbah, C., Malabiau, R.,
Debouzy, J.C., 2000. Nonthermal Effects o f Continuous 2.45 GHz Microwaves on
Fas-induced Apoptosis in Human Jurkat T-cell Line. Bioelectrochemistry and
Bioenergetics. 51:157-161.
24-Porcelli, M; Cacciapuoti, G; Fusco, S; Massa, R; d ’Ambrosio, G; Bertoldo, C; De
Rosa, M; Zappia, Y. 1997. Nonthermal effects o f microwaves on proteins:
thermophilic enzymes as model system. FEBS Lett. 402(2-3):102-6.
2 5-Petersen, B.S., Fojan, P., Petersen, E.I. and Petersen, M.T.N., 2001. The thermal
stability o f the Fusarium solani pisicutinase as a function o f pH. Journal o f Biomed
Biotechnol. 1(2): 62-69.
2 6 -Sato, S., Shibata, C. and Yazu, M., 1996. Nonthermal Killing Effect o f Microwave
Irradiation. Biotech. Tech., 10, 145-150.
•
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27- Steel, B.C., Bilek, M.M., McKenzie, D.R., dos Remedios, C.G., 2002. A technique
for microsecond heating and cooling o f a thin biological sample. Eur Biophys J 31
378-382.
28-Tajchakavit, S. and Ramaswamy, H.S., 1997. Thermal vs. Microwave Inactivation
Kinetics of Pectin Methylesterase in Orange Juice under Batch Mode Heating
Conditions. Lebensm. Wiss. U. Technol., 30, 85-93.
29-Tajchakavit, S. and Ramaswamy, H.S. and Fustier, P. 1998. Enhanced Destruction
o f Spoilage Microorganisms in Apple Juice during Continuous Flow Microwave
Heating. Food research international, Issue 10 , 31, 713-722.
3 0 -Taylor, L.S., 1981. The Mechanisms o f Athermal Microwave Biological Effects.
Bioelectromagnetics 2, 259-267.
3 1 -Tong, Z., 2002. Evaluation o f conventional and microwave heating system for food
processing based on TTI kinetics. A thesis in D ep.of Food Science and
Agr.chemisty, McGill University, Canada. Page: 1-40, 68-95.
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CHAPTER 3
Nonthermal Effects o f Microwave Irradiation on the Structure o f
P-lactoglobulin under Different Physicochemical Conditions
3.1 A BSTRACT
Fourier transform infrared spectroscopy (FTIR) was employed to investigate the
nonthermal effects o f microwave irradiation on the conformation o f P-lactoglobulin in
solution under a variety o f physicochemical conditions. The 1692 cm ' 1 band in the amide
I absorption region in the FTIR spectrum o f P-lactoglobulin was previously assigned to
antiparallel P-sheets hidden in the interior o f the protein. The drop in the intensity o f the
1692 cm ' 1 band is correlated to increased H-D exchange resulting from partial unfolding
or relaxation o f the p-lactoglobulin in solution. Accordingly the band serves as a marker
of the integrity o f the tertiary structure o f the protein. The effect o f multiple-cycle
microwave irradiation at 2450 MHz at 35°C on the intensity o f the 1692 cm ' 1 was
compared to a control set o f p-lactoglobulin solutions heated at the same temperature. In
all case isothermal microwave irradiation resulted in a marked increase in H-D exchange
compared to conventional heating at the same temperature. Heating P-lactoglobulin
solutions at 75 °C by microwave irradiation and conventional heating revealed substantial
changes in the secondary structure o f P-lactoglobulin; however, microwave irradiation
resulted in more extensive aggregation o f the protein in comparison to conventional
heating. A possible mechanism o f the nonthermal microwave effect on P-lactoglobulin
may involve the direct interaction o f microwave energy with the protein backbone leading
to loosing o f the tertiary structure. At higher irradiation temperatures this may enhances
intermolecular entanglement between p-lactoglobulin yielding extensive aggregation.
The nonthermal effect o f microwave was also dependent on the overall charge on Plactoglobulin (by adjusting pH).
pH < p i o f the protein resulted in diminishing the
microwave effect while pH > pi amplified the nonthermal effect o f microwave
irradiation.
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3.2 INTRODUCTION
A body o f scientific evidence supports the existence o f nonthermal effects of
microwaves irradiation (Frohlich, H., 1980, Cleary, S.F., 1989, Marani, E., and
Feirabend, H.K.P. 1994, kirschvink, J.L., 1996, porcelli et a l, 1997, Bohr, H., et al.,
1997, 2000). Thermal effects o f microwave irradiation are related to the heat generated by
the absorption o f microwave energy by water, organic molecules or ions (Porcelli et al.,
1997). The nonthermal effect refers to observable phenomena which do not involve
significant rise in temperature. At the present time, the origin o f nonthermal effects is not
very clear, but it has been postulated to involve direct energy transfer from the
electromagnetic field to the vibrational modes o f macromolecules, altering their
conformation (Taylor, 1981). To date very little experimental evidence is available to
delineate a mechanism o f action o f the putative nonthermal effects. Nonthermal effects
have been reported to activate heat shock response to repair damage caused by partial
unfolding of proteins within cells (Steel, B.C., et al., 2002), microwave heating is
significantly more efficient in inactivating pectin methylesterase (PME) in orange juice
than conventional heating method (Tajchakavit et al., 1997).
The crystal structure o f P-lactoglobulin (Figure 1) reveals the existence of nine
antiparallel 13-strands and one a-helix (Papiz, et al., 1686, Sawyer, 1987, Forge et al.,
2000). In general, all transitions that take place between pH 2 and pH 9 do not cause any
appreciable changes in the native-like p-barrel conformation o f P-lg (Blanch, et al.,
1999). Boye et al. carried out an extensive investigation o f heat-induced unfolding o f Plg in solution as a function o f varying physicochemical conditions such as pH, ionic
strength and in the presence o f different sugars. They observed that P-lg is resistant
unfolding at lower pH and denatured at lower temperature at higher pH. The rates at
which H-D exchange o f amide linkages in proteins undergo isotopic exchange when a
protein is exposed to D 2 O depend on whether the amide N -H moieties are accessible to
the solvent. Hence, N-H exchange rates can be employed as a probe o f protein
conformation and dynamics (Smith, et al., 1997).
The 1692 cm ' 1 band in the FTIR
spectrum o f P-lg has been assigned in to C (0)-N -H amide groups of antiparallel p-
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sheet(s), hidden in the interior o f the protein which undergo very slow H-D exchange at
ambient temperatures (Boye et al., 1995). The intensity o f the 1692 cm ' 1 band was found
to decrease at temperatures well below the denaturation temperature o f p-lg.
Accordingly, the change in the intensity o f this band serves as a probe o f the changes in
the tertiary structure that allow D 2 O to enter the interior o f the protein. The drop in the
intensity of the 1692 cm ' 1 band with increasing number o f heating and irradiation cycles
at a target temperature help delineate the putative nonthermal effect o f microwave
irradiation. While many researchers have disputed the existence o f nonthermal effects of
microwave (Grecz et al., 1964, Olsen et al., 1966; Chipley et al., 1980; Dreyfuss and
Chipley, 1980; Khalil and Villota, 1985) others have provided tentative evidence that
microwave irradiation has nonthermal effect (Frohlich, h 1980, Marani E et al., 1994,
Porcelli et al., 1997, Bohr H et al., 1998, 2000). Bohr et al., (1997, 2000) reported that
microwave irradiation o f cold denatured P-lactoglobulin enhanced the re-folding o f the
protein, while at a higher temperature (48°C) the denaturation o f the protein from its
folded state was enhanced. They proposed that the microwave effect is nonthermal, and
that protein unfolding takes place when the amplitude o f a wring excitation becomes so
large that it is energetically favorable to bend the protein backbone. Wring modes o f
proteins o f specific lengths can resonate with molecular modes o f the protein. In this
work FTIR spectroscopy is employed to examine the nonthermal effects o f microwave
irradiation on the secondary and tertiary structures o f P-lactoglobulin under varying
physicochemical conditions.
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Figure 3.1. The crystal structure o f a single subunit o f bovine f - l g (Brownlow et al.,
1997).
3.3 M aterials and M ethods:
Bovine P-lactoglobulin contained an equal mixture o f te two genetic variants A
and B (p-lg) was a generous gift from Davisco Foods International (MN, USA). The purty
of the protein was confirmed by gel electrophoresis, spectroscopy, ESI-MS, FT-Raman
and FTIR spectroscopy. D 2 O was obtained from Aldrich (Milwaukee, WI, 53233, USA).
To study the effect o f pH on a protein that subjected to microwave irradiations, P-lg (5%
w/v) was dissolved in 0.2M deuterated phosphate buffer o f pH 2, pH 7, and pH 9. (In this
work, pH is employed in place o f pD, and calculated from pD = pH + 0.4 (Boye, et al.,
1995). p-lg (5% w/v) solution where also prepared in deuterated solutions of 0.5 M and 2
M NaCl. Two sets o f p-lg solutions were prepared in triplicates, (1 ml p-lg 5% w/v
solution in each tube) each triplicate set was employed in the microwave irradiation and
conventional heating studies. The temperature was measurements were within + 0.5 °C.
3.3.1 M icrowave T reatm ents:
P-lg solutions prepared above were subjected to microwave irradiation using a
focused microwave Synthewave 402 (PROLABO, 54, rue Roger Salengro - BP 115 94126 FONTENAY-SOUS-BOIS CEDEX), operating at a frequency o f 2.45 GHz (k = 12
cm) at power levels between 15-300 Watts and a temperature range between 0 and 450°C
with a magnetron overheating safety system.
The following parameters were employed to irradiate the p-lg solutions:
Fixed power: 15 W (5 % o f the total power) or 48 W (16 % o f the total power), depending
on the pH and desired temperature. In the first group o f experiments;
samples were
irradiated until 35°C (40 seconds for p-lg in D 2 O (pH ~ 6 .8 ), 22 seconds for P-lg solutions
at pH 2, 17 seconds for p-lg solutions at pH 7, and 13 seconds for P-lg solutions at pH 9.
To study the effect o f microwave irradiation on the rate o f aggregation, P-lg solutions (5
% w/v in D2 O) were subjected to microwave irradiation until 75°C (70 seconds at 48
Watts).
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Microwave irradiation was applied to P-lg solutions for 1-10 cycles (in each cycle, the
samples were subjected to microwave irradiation until it reached the desired temperature
(35 or 75)°C, then the samples were immediately placed in a cold water bath until they
reached 4°C from 35 °C, or to 20 °C from 75 °C.
3.3.2 C onventional H eating T reatm ents:
P-lg solutions (5% w/v in D 2 O) were heated in a water bath matching the
irradiation times.
3.3.3 F o u rier T ransform In fra re d (FTIR ) Spectroscopy:
FTIR spectra were recorded (512 scans at 4 cm '1) on a Magna 550 (Nicolet,
Madison, WI) equipped with a DTGS detector and purged with dry air. A sample volume
of
8
pi was placed in an IR cell consisting o f two CaF 2 windows separated by a 50 pm
spacer.
The temperature o f the sample was regulated by placing the IR cell in a
temperature-controlled holder. The reported temperatures are accurate to within + 0.1 °C.
Resolution enhancement o f the spectra were achieved with the use o f the Fourier self­
deconvolution (FSD ) function in OMNIC
6
(Nicolet, Madison, WI) employing a
bandwidth o f 20 cm '1, and enhancement factor (k) o f 2.4 as described by Kauppinen et
al., 1981. The peak heights o f the amide I’ bands were normalized by dividing each peak
height by the area o f the entire amide I bands between 1700 and 1600 cm ' 1 (after baseline
correction o f the same spectral region). All samples were measured in triplicates and
statically analyzed using Origin (Microcal Software Inc, MA). The standard deviation
between triplicate measurements was ~ 3 %.
3.4 RESULTS
The deconvoluted FTIR spectrum o f P-lactoglobulin in D 2 O in the amide I
between 1700 and 1600 cm '1, at 25°C is shown in Figure 3.2 Six bands can be clearly
discerned and have been previously assigned by others (Susi and Byler, 1983, Casal et al.,
1988, Byler and Farrell, 1989, Boye et al., 1995) as follows: 1692 cm ' 1 is a high
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frequency band o f antiparallel [3-sheets, hidden in the interior o f the protein that have not
undergone H-D exchange (Boye et al., 1995), the 1682 and 1676 cm ' 1 bands are assigned
to high frequency bands of antiparallel P-sheets, that have undergone H-D exchange, the
band at 1662 cm ' 1 is assigned to turn structures while the band at 1647 cm ' 1 is assigned to
an a-helix. The bands at 1634 cm ' 1 is assigned to the low frequency band o f antiparallel
P-sheets while the band at 1622 cm ' 1 is attributed to extended P-sheet structure.
Antiparallel
P-sheet
3.0
2.8
2.6
2.4
P-Sheet
2.2
a-Helix
2.0
O
(A
A
P-Turns
<
0.8 -|
Antiparallel
P-sheet
0 6 I Antiparallel
0 4 | p-structure
02
\
o.o
\s£Z/Z.TfTTL.
1700
1680
1660
1640
1620
W avenumbers (cm'1)
Figure 3.2 The amide I region o f the FTIR spectrum o f P-lactoglobulin in D 2 O (5% w/v)
at 25 °C after Fourier self-deconvolution (bandwidth = 20 cm'1 and k factor o f 2.4).
»
33
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3.4.1 Effect o f Microwave Irradiation on the Structure o f B-lg:
Figure 3.3 shows an overlaid plot o f the amide I bands in the FTIR spectra o f |3-lg
solutions as a function o f increasing microwave irradiation cycles at 35 °C. The intensity
o f the 1692 cm '1 band decreases with increasing number o f cycles. The 1692 cm '1 band
represents the extent o f H-D exchange o f the solvent-inaccessible P-sheets in the interior
o f p-lg. The intensity o f this band decreases when P-lg solution is heated by either
microwave irradiation (Figure 3.4) or by conventional heating (Figure 3.5).
3.0
2.8
2.6
2.4
2.2
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2.0
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0.8
0.6
Increasing
Cycles 1-10
0.4
0.2
0.0
1700
1680
1660
1640
1620
W avenum bers (cm '1)
Figure 3.3 Overlaid FTIR spectra in amide I absorption region o f fi-lg solutions in D 2 O
subjected to 1-10 cycles o f microwave irradiation at 35°C. A ll spectra were recorded at
25°C.
34
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Increasing number
of cycles 1-10
1
0.22
0.20
0.18
oo>
0.16
c
0.14
JQ
0.12
ra
O
(0
JQ
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0.10
0.08
0.06
0.04
0.02
0.00
1700
1698
1696
1694
1692
1690
1688
Wavenumbers (cm'1)
Figure 3.4 A plot o f the changes in the intensity o f the 1692 c m 1 band in the FTIR
spectra o f P-lg in D 2 O as a function o f increasing irradiation cycles at 35 °C ( ---------- )
represents the change in the band intensity after 10 irradiation cycles.
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Increasing number
of cycles 1-10
i
0.24
0.22 i
0.20 1
0.18 1
0.16 1
0o>
c
n(0
i.
o
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<
0.14 1
0.12 1
0.10 1
0.08
0.04 1
0.02 i
1700
1698
1696
1694
1692
1690
1688
Wavenumbers (cm )
Figure 3.5 A p lo t o f the changes in the intensity o f the 1692 c m 1 band in the FTIR
spectra o f fi-lg in D 2 O as a function o f increasing heating cycles at 35 ° C ( -------- )
represents the change in the band intensity after 10 heating cycles.
Comparison o f the extent of the changes in the intensity o f the 1692 cm '1 band as
a function o f increasing irradiation and heating cycles reveals that the differences between
the microwave irradiation effect and the conventional heating effect on the intensity o f
the 1692 cm '1 band is more pronounced with increasing number o f irradiation cycles.
Only a small loss in the intensity o f the 1692 cm '1 band is observed by conventional
heating with increasing number o f cycles. An 11 % drop in the intensity o f the 1692 cm '1
band is observed when the P-lg solutions in D 2 O is left at 25 °C for 7 hours (the total time
required to complete the heating or microwave irradiation experiments).
Both microwave irradiation and conventional heating experiments have the same
effect on the intensity o f the 1692 cm’1 band after one treatment cycle. A difference in
the treatment method starts to appear after the second cycle, with larger differences
36
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observed with increasing number o f cycles. The intensity o f the 1692 cm' drop by 50%,
and 78% after 4th and 10th cycles respectively as a result o f microwave treatment
compared to 7% and 25% drop respectively by conventional heating under the same
conditions (Figure 3.6).
450
400
350
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Figure 3.6 A plo t o f the drop in the pea k height o f the 1692 c m 1 band in the FTIR
spectrum o f f - l g in D 2 O as a function o f microwave irradiation (black) and conventional
heating (white) at 35°C.
37
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3.4.2 Effect o f Microwave Irradiation on B-lg solutions at Different pHs
The FTIR spectra o f P-lg in D20-phosphate buffer (pH 2, 4, 7 and 9) in the amide
I’ absorption region are shown in Figures 3.7(A+B). The effect o f pH on the structure o f
P-lg has been studied by Boye et al., 1995.
2.8
2.6
2.4
2.2
2.0
0.6
0.4
0.2
0.0
1700
1680
1660
1640
1620
Wavenumbers (cm'1)
Figure 3.7A. The amide I region o f FTIR spectra o fft-lg at different pHs. (— ) represent
P-lg in p H 7, ( ......) represent (3-lg in p H 2, (- ■- ) represent P-lg in p H 4. (— ■-) represent
jl-lg in p H 9
38
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4.2
4.0
3.8
3.6
3.0
■}
pH
2.8 4
CJ
o 2 41
8 2.2 i
8
pfi 2.0 -I
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09
pfi
1.6
1.4
0.8
0.6
0.4
0.2
0.0
1700
1680
1660
1640
1620
Wavenumbers (cm 1)
Figure 3 .7B. The amide I region o f FTIR spectra o fP -lg in different pH s as indicated on
the graph.
39
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3.4.2.1 E ffect o f microwave irradiation on the Ji-lg at p H 2
Changes in the intensity o f the 1692 cm '1 band o f P-lg in solutions at pH 2
subjected to microwave irradiation or conventional heating are shown in Figure 8. The
results reveal that the intensity o f the 1692 cm '1 band drops more significantly in
microwave irradiated samples compared to conventional heat treatment. The 1692 cm '1
band loses about 40 % o f its intensity after 10 cycles o f microwave irradiation compared
to 78 % loss in P-lg solution irradiated in D 2 O at 7 pH solutions under the same
conditions.
o>
300
3
4
5
6
7
8
Number of Cycles
9
10
N2
Figure 3.8 A p lo t o f the change in the peak height o f the 1692 c m 1 in the FTIR spectra
o f P-lg in D 2 O at p H 2 at 35°C as a function heating cycles by microwave irradiation
(black) or conventional heating (white) .
40
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3.4.2.2 The effect o f microwave irradiation on the p-lg at p H 4
Only a very slight decrease in the intensity o f the 1692 cm '1 band in the FTIR
spectrum o f P-lg was observed at pH 4 as a function o f heating cycles. The microwave
effect was still a little more pronounced compared to conventional heating (Figure 3.9).
The intensity o f the 1692 cm '1 band dropped only 26 % after 10 cycles o f microwave
irradiation compared to 7 % drops after 10 cycles o f conventional heating.
4499^8
S> 250
4
5
6
7
10
N2
N um ber of Cycles
Figure 3.9 A plo t o f the change in the pea k height o f the 1692 c m 1 in the FTIR spectra
o f P-lg in D 2 O at p H 4 at 35°C as a function heating cycles by microwave irradiation
(black) or conventional heating (white) .
41
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3.4.2.3 The effect o f microwave irradiation on the fi-lg at p H 9:
Approximately 88% drop in the intensity o f the 1692 cm '1 band is observed after
10 cycles o f microwave irradiation compared to a slight decrease (16 %) after 10 cycles
of conventional heating (Figure 3.10).
N1
1
3
4
5
6
7
Number of Cycles
Figure 3.10 A p lo t o f the change in the peak height o f the 1692 c m 1 in the FTIR spectra
o f fl-lg in D 2 O at p H 9 at 35°C as a function heating cycles by microwave irradiation
(black) or conventional heating (white) .
42
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3.4.3 The Effect of Microwave Irradiation on the B-Ig in 0.5 M NaCl Solution:
Subjecting P-lg to microwave irradiation in 0.5 M NaCl solutions, resulted in an
89 % drop in the intensity o f the 1692 cm '1 band after 10 cycles compared to a 15 % drop
by conventional heating after 10 cycles (Figure 3.11).
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1
3
4
5
6
7
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Figure 3.11 A Plot o f the change in the peak height o f the 1692 c m 1 in the FTIR spectra
o f fl-lg in 0. 5 M NaCl in D 2 O at 35°C as a function heating cycles by microwave
irradiation (black) or conventional heating (white) .
43
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3.4.4 The Effect of Microwave Irradiation on the B-Ig in 2 M NaCl Solution
The 1692 cm '1 band lost about 78% o f its intensity after 10 cycles o f microwave
treatment compared to 8% loss when P-lg was heated under the same conditions
(Figure 3. 12).
£
<D
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250
150
N1
1
2
3
4
5
6
7
8
9
10
N2
Number of Cycles
Figure 3.12 A Plot o f the change in the peak height o f the 1692 c m 1 in the FTIR spectra
o f P-lg in 2 M NaCl in D 2 O at 35°C as a function heating cycles by microwave
irradiation (black) or conventional heating (white) .
44
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3.4.5 Comparisons between Multiple Heating Cycles at 75 °C and Multiple
Microwave Irradiation Cycles (at the same temperature) o f B-lg Solutions:
The FTIR spectra in the amide I absorption region o f (3-lg in D 2 O subjected to
microwave irradiation to reach at temperature o f
75°C as a function o f increasing
number o f cycles (1, 3, 5, 7 and 10) are shown in Figure 3.13. The bands at 1684 and
1615 cm’1 are attributed to intermolecular hydrogen-bonded antiparallel p-sheet sheets
(Ismail et al., 1992) formed when the protein undergoes irreversible aggregation. The
intensity of the two aggregation bands increases markedly with increasing number of
irradiation cycles. The 1692 cm '1 disappeared completely after one microwave irradiation
cycle. Figure 13 also shows a decrease in the intensity and the broadening o f the amide I’
bands of p-lg. The band at 1647 cm’1 which has been attributed to a-helical structure
(Casal et al., 1988; Susi and Byler, 1988) shifted to 1645 cm’1 after one irradiation cycle.
The intensity o f the 1647 cm '1 band also decreased dramatically from 3 to 9 cycles and
disappeared after 10 irradiation cycles. A significant decrease in the intensity o f the 1633
cm’1band (assigned to antiparallel P-sheets) was observed with a shift from 1633 to 1628
cm’1 with increasing cycles o f microwave irradiation. An initial increase in intensity o f
the 1622 cm '1, attributed to P-sheet structure, was observed after one irradiation cycle, the
band disappeared after the 3rd irradiation cycles. By comparison, P-lg in D 2 O subjected to
conventional heating to 75 °C under the same conditions o f the microwave irradiation
experiment, showed very slight increase in the intensity o f the aggregation bands at 1682
and 1615 cm '1. Only a shift in the 1676 cm’1 band to 1674 cm '1 was observed after 10
cycles o f conventional heating. The 1647 cm '1 band shifted to 1645 cm '1 after one cycle,
and remained at 1644 cm '1 with a slight decrease in its band intensity. A substantial
decrease in the band intensity o f 1633 cm '1was observed after heating p-lg till 75°C with
a shift to 1631 cm '1 after one cycle, and 1629 cm '1 after 5 heating cycles. A smaller
increase in the intensity at 1622 cm '1was observed between the 1st and 3r heating cycles
and disappeared after 5 heating cycles (Figure 3.14). Figure 3.15 shows the change in the
peak height of the 1615 cm '1 aggregation in the FTIR spectra o f p-lg solutions heated to
75 °C as a function o f the number o f heating and irradiation cycles.
•
45
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0.28
0.26
0.24
In c re a s in g
num ber of
c y c le s
0.22
0.20
0.18
0)
o
c
<0
0.16
O
(0
0.10
.Q
u.
.a
<
0.14
0.12
0.08
0.06
0.04
0.02
0.00
- 0.02
1700
1680
1660
1640
1620
Wavenumbers (cm )
Figure 3.13 Overlaid FTIR spectra in the amide / ’ absorption region o f fi-lg solutions
subjected to microwave irradiation to 75°C fo r 1, 3, 5, 7, and 10 cycles (70 seconds o f
irradiation at 48 W per cycle and a cool down period to room temperature o f 5 minutes
46
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0.20
CL)
£C=J
0.15
C*3
£3
o
CO
.Q
<
Increasing
cycles
0.10
0.05
0.00
1700
1680
1660
1640
1620
W avenum bers (cm-1)
Figure 3.14 Overlaid FTIR spectra in the amide / ’ absorption region o f f- lg solutions
subjected conventional heating in a water bath until the temperature reaches 75°C fo r
1, 3, 5, 7, and 10 cycles (70 seconds o f heating per cycle and a cool down period to room
temperature o f 5 minutes between each cycles).
47
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Num ber of Cycles
Figure 3.15 A Plot o f the change in the peak height o f the 1615 c m 1 in the FTIR spectra
o f [1-lg in D 2 O at heat to 75°C as a function heating cycles by microwave irradiation
(black) or conventional heating (white) .
48
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3.5 DISCUSSION
Figures 3.6 to 3.10 clearly demonstrate the rate o f increase o f H-D exchange in (3lg is far more sensitive to microwave irradiation than conventional heating under similar
physiochemical conditions. The effect o f microwave irradiation is more pronounced at
higher pH indicating that the microwave interaction with P-lg in solution is more
efficient. The microwave effect on the rate o f H-D exchange in 0.5 M NaCl solution of
P-lg was higher than the microwave effect on P-lg in D 2 O and in 2 M NaCl solution.
However the microwave effect was still more pronounced than the conventional heating
effect on the rate o f H-D exchange under the same conditions. This suggests that P-lg was
more stable in D 2 O and 2 M NaCl solution than it was in 0.5 M NaCl solution,
accordingly, this could not be explained in terms o f the unavailability o f D2 O molecules
for protein salvation at higher salt concentrations but rather to possible perturbation of
salt bridge interactions by low concentrations o f Na+ and C f ions. The effect of
temperature on P-lg at different pH values was examined by Boye et al., 1995. They
reported that the 1692 cm '1 band disappeared when the P-lg solutions were heated to 72,
59, 51, and 47°C for pH 3, 5, 7, and 9 respectively. This meant that P-lg was in a more
relaxed state at pH 9 and in a more compact state at lower pH.
Boye et al., 1995 using
variable-temperature FTIR spectroscopy, and DSC analysis observed only minor changes
in the secondary structure o f B-lg dissolved in 0.5 to 2 M NaCl solutions after 20 h at
25°C. P-lg in D 2 O at different pH, aggregated faster when its exposed to microwave
irradiation compared to exposure to conventional heating under the same conditions.
The nonthermal microwave effect increased gradually with increasing number o f
irradiation cycles. Similar effects were reported upon irradiation o f P-galactosidase at
70°C where a more irreversible inactivation o f the enzyme was achieved compared w to
conventional heating at the same temperature (F.la Cara et al,. 1999). Bohr et al,. 1999
also postulated that microwave radiation may speed up rates o f folding and unfolding o f
globular proteins in solutions.
Recently, David et al., 2003 reported that exposure of
BSA to microwave irradiation enhanced protein aggregation.
49
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3.6 PROPOSED MECHANISM
Although the mechanism of the nonthermal effect is unknown, violent motion o f
dipoles in molecules by microwave field seems to destroy structures o f polyatomic
molecules such as protein and phospholipids. Microwaves either cause ions to accelerate
and collide with other molecules or cause dipoles to try to rotate and line up with rapidly
alternating (2,450 million times per second) electrical field (Sato, S. et al., 1996).
Aytekin, C. investigated the effects o f 2450 MHz microwave on the activity of
alpha-amylase. They found that the activity o f this enzyme depended on the power
density and the exposure time to microwave. They proposed that the deformation tertiary
structure o f protein originates from changing rotation surrounding C a-C and C a-N
bonds. They concluded that protein chains were broken by microwave depended on
power level (Aytekin, C. et al., 2003).
Our results reveal that microwave treatment accelerated the rate o f H-D exchange.
This may be attributed to a direct energy transfer from the electromagnetic field to the
vibrational modes o f protein, resulting in an increase in displacement o f the protein
backbone (Taylor, 1981) and resulting in increased intra-domain motion which allows the
solvent to penetrate deeper into the protein interior resulting in increased H-D exchange.
The increase in the intra-domain motion coupled with loosening or weakening the
secondary structure of the protein at the Tm could result in increased entanglement leading
to an increase in aggregation o f P-lg.
Altering the charge balance on the protein (by adjusting pH) is known to effect
domain-domain interactions. This will result in enhanced or diminished motion between
the domains. An increase in overall positive charge, by reducing the pH below its
isoelectric point (5.3), has been shown to stabilize P-lg against unfolding and aggregation
possibly by increasing the number o f salt bridges. This leads to a reduction in intra­
domain motion. While an increase in the overall negative charge (pH > PI) results in
domain-domain repulsion leading to increased intra-domain motion leading to an increase
in H-D exchange under conventional heating.
However, the interaction o f the unencumbered domains with electromagnetic field
o f the microwave at pH > p i can result in a marked increase in domain angulations. This
50
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leads to an enhanced H-D exchange at lower temperatures and increased entanglement
resulting in extensive aggregation near the Tm o f the protein.
51
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•
REFERENCES
----------------------------------------------------------------
1- Aytekin, C., Dasdag, S., Gulsun, I., Ensari, Y., Kaya, Z., 1995. Effects of
Microwave radiation on Bacterial a-amylase Activity. J. F ood Sciences and
Technologies 3nr. 28-30.
2- Bohr, H. and Bohr, J., 2000. Microwave-Enhanced Folding and denaturation of
globular proteins, the American Physical Society, V 61, number 4.
3- Bohr, H. and Bohr, J., 2000. Microwave enhanced kinetics observed in ORD studies
o f a protein. Bioelectromagnetics 21:68-72.
4- Bohr, H., Bohr, J., Brunak, S., 1997, Protein Folding and Wring Resonances.
Biophysical Chemistry 63, 97-105.
5- D ’Alfonso, L., Collini, M. and Baldini, G., 2002. Does (3-lactoglobulin Denaturation
Occur via an Intermediate State? Biochemistry 41, 326-333.
6- Decareau, R.V, 1985. Microwave in the Food Processing Industry. Text book.
TP370.5.D43 ISBN 0-12-208430-6, Academic Press, Inc. Orlando, Florida. P 1-37
7- De Pomerai, D., Daniells, C., David, H., Allan, J., Duce, I., Mutwakil, M., Thomas,
D., Sewell, P., Tattersall, J., Jones, D., Candido, P., 2000, Cell biology: Nonthermal
heat-shock Response to Microwaves. Nature 405, 417-418.
8- De Pomerai, D.I., Smith, B., Dawe, A., North, K., Smith, T., Archer, D.B., Duce,
I.R., Jones, D., Peter, E., Candido, M., 2003. Microwave Radiation can Alter
Protein Conformation without Bulk heating. FEBS Letters 543, 93-97.
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8- Dreyfuss, M. S. and Chipley, J.R., 1980. Comparison o f Effects o f Sublethal
Microwave Radiation and Conventional Heating on the Metabolic Activity of
Staphylococcus Aureus. Appl. Microb. 39(1): 13-16.
9- Englander, J.J., Mar, C.D., Li, W., Englander, S.W., Kim, J.S., Stranz, D.D.,
Hamuro, Y. and Woods V.L., 2003. Protein Structure Change Studied by hydrogendeuterium Exchange, Functional Labeling, and Mass Spectrometry. PNAS,
june 10,2003, Vol.100, no. 12, P 7057-7062.
10-Englander, S.W., and Krishna, M.M.G., 2001. Hydrogen Exchange.
Nature Structural Biology 8 1-2.
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11-Hoedemaeker, F.J; Visschers, R.W; Alting, A.C; de Kruif, K.G; Kuil, M.E and
Abrahams, J.P, 2002. A novel pH-dependent Dimerization M otif in P-lactoglobulin
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pig
(Sus
scrofa).
Acta
Crystallographica,
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Biological
Crystallography D58, 480-486.
12-Ipsita, R.; and Munishwar, N.G., 2003. Nonthermal Effects o f Microwaves on
Protease-catalyzed Esterification and transesterification. Tetrahedron 59, 5431-5436
13-Kaufmann, B and Christen, P 2002. Recent Extraction Techniques for Natural
Products: Microwave-assisted Extraction and Pressurized Solvent Extraction
Phytochemical Analysis. V.13, 105-113.
14-Kinetics o f Microbial Inactivation for Alternative Food Processing Technologies,
Microwave and Radio Frequency Processing. U.S Food and Drug Administration,
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for
Food
Safety
and
Applied
Nutrition,
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2,
2000.
http://vm.cfsan.fda.gov/~comm/ift-micr.html
15-Koutchma, T., LeBail, A. and Ramaswamy, H.S, 2001. Comparative Experimental
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o f Microbial
Destruction
in
Continuous-flow
Microwave
and
Conventional Heating Systems. Canadian Biosystems Engineering 43, 3.1-3.8.
16-La Cara, F., Scarfi, M.R., D ’Auria, S., Massa, R., D ’Ambrosio, G., Franceschetti,
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Conventional Heat on the Activity o f a thermophilic P-galactosidase from Bacillus
acidocaldarius. Bioelectromagnetics 20:172-176.
17-Laurence, J.A.; French, P.W.; Lindner, R.A. and McKenzie, D.R., 2000. Biological
Effects o f Electromagnetic Fields-Mechanisms for the Effects o f Pulsed Microwave
Radiation on Protein conformation. J. Theor. Biol. 206, 291-298.
18-Monahan, F.J., German, B. and Kinsella, E., 1995. Effect o f pH and Temperature on
Protein Unfolding and Thiol/Disulfide Interchange Reactions during Heat-Induced
Gelation o f Whey Proteins. J. Agric. Food Chem. 43, 46-52.
19-Mudgett, R.E., 1986. Microwave Properties and Heating Characteristics o f Foods
Food Technol. 40, 84-93.
2 0-Peinnequin, A., Piriou, A., Mathieu, J., Dabouis, V., Sebbah, C., Malabiau, R.,
Debouzy, J.C., 2000. Nonthermal Effects o f Continuous 2.45 GHz Microwaves on
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Fas-induced apoptosis in Human Jurkat T-cell line. Bioelectrochem. Bioenerg.
51:157-161.
21-Porcelli, M; Cacciapuoti, G; Fusco, S; Massa, R; d ’Ambrosio, G; Bertoldo, C; De
Rosa, M; Zappia, Y. 1997. Nonthermal effects o f microwaves on proteins:
thermophilic enzymes as model system. FEBS Lett. 402(2-3): 102-6.
22-Ptak, D, 1998. Rigid-body oscillations o f a-helices: implications for Protein
Thermal Stability. Biophysical Chemistry 73, 121-127.
23- Sato, S., Shibata, C. and Yazu, M., 1996. Nonthermal Killing Effect o f Microwave
Irradiation., Biotech. Tech., 10, 145-150.
2 4 -Smith, L., Deng, Y. and Zhang, Z., 1997. Probing the Non-covalent Structure of
Proteins by Amide Hydrogen Exchange and Mass Spectrometry. Journal o f Mass
Spectrometry, 32, 135-146.
25- Steel, B.C., Bilek, M.M., McKenzie, D.R., dos Remedios, C.G, 2002. A technique
for Microsecond Heating and Cooling o f a thin Biological Sample. Eur Biophys J
31 378-382.
2 6 -Tajchakavit, S. and Ramaswamy, H.S., 1997. Thermal vs. Microwave Inactivation
Kinetics o f Pectin Methylesterase in Orange Juice under Batch Mode Heating
Conditions. Lebensm. Wiss. U. Technol., 30, 85-93.
2 7 -Tajchakavit, S. and Ramaswamy, H.S. and Fustier, P 1998. Enhanced Destruction
o f Spoilage Microorganisms in Apple Juice during Continuous Flow Microwave
Heating. Food research international, Issue 10 , 31, 713-722.
28-Taulier, N and Chalikian, T.V, 2001, Characterization o f pH-induced Transitions of
P-lactoglobulin: Ulterasonic, Densimetric, and Spectroscopic Studies. J.Mol.Biology
314,873-889.
2 9 -Taylor, L.S., 1981. The Mechanisms o f Athermal Microwave Biological Effects.
Bioelectromagnetics 2, 259-267.
30-Ugolini, R; Ragona, L; Silletti, E; Fogolari, F; Visschers, R.W; Alting, A.C and
Molinari, H, 2001. Dimeriaztion, Stability and Electrostatic Properties o f Porcine plactoglobulin J.Biochemistry 268, 4477-4488.
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3 1 -Tong, Z., 2002. Evaluation o f Conventional and Microwave Heating System for
Food Processing based on TTI kinetics. A thesis in Department o f Food Science and
Agr.chemisty, M cGill University, Canada. Page: 1-40, 68-95.
55
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CHAPTER 4
The Unfolding Pathway o f P-Lactoglobulin under
Microwave/conventional Heating Treatment
4.1 ABSTRACT
The unfolding pathway o f P-lactoglobulin (P-lg), exposed to microwave and
conventional heating was examined by Fourier transform infrared (FTIR) spectroscopy,
and two-dimensional (2D) IR correlation spectroscopy. Conventional heating cycles from
ambient temperatures to 35°C and by microwave irradiation cycles to the same
temperature of P-lg solution were carried out in D 2 O. The pre-heated samples were then
examined by variable-temperature FTIR spectroscopy to produce a series o f dynamic
spectra. Examination o f the synchronous and asynchronous maps generated from the
dynamic spectra revealed the sequence o f unfolding pathways o f conventionally heated
and microwave heated P-lg solutions were comparable. The experimental data also
revealed that the process leading to aggregate formation and gelation o f the protein
started with an increase in the random coil structures followed by the loss o f p-sheets and
a subsequent loss o f a-helical conformations. The final event was the formation of
intermolecular P-sheets. However, the rate o f protein unfolding and aggregate formation
was substantially higher in the microwave treated samples.
56
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4.2 INTRODUCTION
Protein folding and unfolding processes are driven mainly by the polarity of
proteins and hydrophobic effects between the protein and surrounding solvent. The
hydrophobic side chains have a propensity to be found at the core o f the majority of
proteins, while at the surface, residues with side chains favor contact with water
(Richards, 1991).
B-lactoglobulin (P-lg) is an excellent model for studying the folding/unfolding
behavior as it is well characterized nature and has a relatively low molecular mass. P-lg
exists at the normal pH o f bovine milk as a dimer (Timasheff and Townend, 1961), with
a molecular weight o f approximately 36 kDa, and consists o f two monomeric subunits
with a molecular weight o f 18350 Da (162 residues). Each monomer is composed o f two
orthogonal slabs o f nine antiparallel P-strands and one a -helix (Papiz et al., 1986,
Monaco et al., 1987). Each B-lg monomer has two disulfide bonds and one free thiol
group (Figure 4.1), which exhibit increased reactivity above pH 7 (Kinsella and White
head, 1989).
The majority o f the solvent-protected amide groups are involved in hydrogen bonding
within the F-G-H m otif and along the hydrophobic face o f the adjacent amphipathic ahelix.
Globular proteins display a wide range o f heat-induced behavior, which reflect
their inherent differences in molecular and physical structures and intermolecular
interactions (Phillips et al., 1994). Thermal denaturation o f B-lg leads to the formation o f
a significant amount o f intermolecular B-structures as the temperature is progressively
increased from 60 to 95°C (Boye et al., 1995). The effect o f microwave and conventional
heating on the unfolding and aggregation of P-lg has been recently investigated by ID
variable-temperature FTIR spectroscopy and revealed the existence o f a nonthermal
microwave effect responsible for an substantial increase in H-D exchange o f amide
groups hidden within the interior o f p-lg and a marked increase in intermolecular P-sheet
of microwave induced denaturation o f P-lg. In this work, the sequence o f event leading to
•
the unfolding of P-lg exposed to conventional and microwave heating is examined by
57
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
two-dimensional IR correlation analysis employing the dynamic spectra generated in our
previous study (Chapter 3).
I Strand
Figure 4.1 Dimer interface o f fi-LG from the X-ray coordinates, 1BEB. Side view (a) and
top view (b) with respect to f i l strands. Close-up views o f AB loops (a) and f i l strands (b)
are also shown, where intersubunit hydrogen bonds between side chains and main chains
can be seen.
58
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.3 Materials and Methods:
P-Lactoglobulin (A + B) was a gift from Daviso Foods International (MN, USA)
and used as is. D 2 O was purchased from Aldrich Inc. (Milwaukee, WI, 53233, USA).
4.3.1 Microwave Treatments:
SYNTHEWAVE 402 microwave (PROLABO, 54, rue Roger Salengro - BP 115 94126 FONTENAY-SOUS-BOIS CEDEX) was employed for microwave treatment of all
samples. The system operates at a frequency o f 2.45 GHz (X = 12 cm) using a focused
microwave with adjustable power between 15-300 Watts, is equipped with a magnetron
overheating safety system and a safety switch on the door which prevents emission of
microwaves and a temperature sensor. The microwave system was controlled by Prolabo
software (PROLABO, 54, rue Roger Salengro - BP 115 - 94126 FONTENAY-SOUSBOIS CEDEX)). The microwave irradiation parameters employed a fixed power of 15 W
or 48 W. Different irradiation times were employed to achieve the desired temperature.
Solutions o f deuterated P-lg were prepared by dissolving P-lg (5 % w/v) in D 2 O
and leaving it for 50h 25°C. Triplicates o f P-lg solution (1 ml in each tube) were placed
in the first group o f experiments in the cavity o f the SYNTHEWAVE 402 microwave,
and irradiated with 15 W till the samples reached 35°C (within 40 seconds).
In the
second group of experiments, of P-lg solutions were subjected to microwave irradiation in
the cavity of the SYNTHEWAVE 402 microwave with power o f 48 W until the samples
reached 75°C (within 70 seconds).
In each set o f experiments the sample was repeatedly heated to the target
temperatures from 1 to 10 cycles. For each cycle, samples were subjected to microwave
irradiation till they reached the desired temperature (35 or 75°C), then the microwave was
turned off, the samples were taken out, and then placed in a cold water bath till they
reached 4°C (to allow longer irradiation times in the case o f heating to 35 °C) or to 25 °C
for samples heated to 75 °C.
•
59
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4.3.2 Conventional Heating Treatments:
P-lg solutions (5% w/v) in D 2 O were conventionally heated in a water bath under
the same conditions as in the microwave experiments, i.e., the target temperature was
reached in the same time it took in the microwave. This had been achieved by adjusting
the initial temperature o f the water bath to be higher than the target temperature.
4.3.3 Fourier Transform Infrared (FTIR) Spectroscopy:
The FTIR spectra were recorded using a 8210E Nicolet spectrophotometer
equipped with a DTGS detector (Nicolet, Madison, WI). The spectrometer was purged
with dry air from a Balston dryer (Balston, Haverhill, MA, 01835-0723, USA). The
temperature o f the sample was regulated by placing the cell in a holder employing an
Omega temperature controller (Omega, Engineering, Laval, QC). A total o f 512 scans at
resolution at 4 cm '1 were co-added o f a 7 pi P-lg solution (5 % w/v), placed between two
CaF 2 windows separated by a 50pm spacer. For variable-temperature measurements, the
temperature o f the protein solution was increased in 5°C increments and allowed to
equilibrate for 10 min prior to data acquisition. The reported temperatures are accurate to
within ± 0.5 °C. Samples were heated till 90°C. Only changes that took place up on
heating till 55°C were selected for this study.
Mathematical resolution enhancement techniques
employing Fourier self­
deconvolution (FSD) was employed to separate amide I band components as described by
Kauppinen et al., 1981, using bandwidth o f 20 cm '1, and an enhancement factor (k) o f 2.4.
The signal to noise ratio was >20,000:1. All the spectra were baseline corrected between
1700-1600 cm '1. Spectra were normalized by dividing each amide I band component by
the integrated area o f the entire amide I band between 1700-1600 cm '1. All FTIR
measurments experiments were done in triplicates, data has been statically analyzed using
Origin (OriginLab Corporation, formerly Microcal Software, Inc. U.S.A), A standard
deviation of 3-4 % between triplicate runs was achieved.
60
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4.3.4 KG2D Software for 2D Correlation Spectroscopic Analysis:
2D correlation analysis was performed using the KG2D software written by
Y.Wang. Perturbation-induced fluctuation o f spectroscopic signals can be o f an arbitrary
form, not limited to a sinusoidal form as required by the original 2D IR correlation
spectroscopy (Noda, 1990). One o f the major features o f the KG2D software in
conjunction with the Grams 3D software (Galactic Inc.) is the ability to plot a threedimensional (3D) representation o f the 2D maps, enabling one to observe subtle
correlation peaks by rotating and enlarging the 3D image. W ith the conventional 2D
contour representation o f 2D correlation spectra, weak peaks are not discerned if the
number o f representation lines is reduced in the 2D contour map. Furthermore, some
“valleys” in the 2D correlation spectrum may be misinterpreted as correlation peaks. By
viewing the horizontal and vertical slice profiles o f 3D spectral surface, the weak peaks
can be accurately determined. The data processing protocol is summarized in Figure 4.2
Raw Data
(spectra)
Base line
correction
Fourier self-deconvolution
Region (1700-1500) cm'1
▼
Synchronous &
Asynchronous
maps
A-------------
2D
correlation
a-------------
Normalization
Figure 4.2 The 2D correlation procedure
4.4 RESULTS
Six major amide I ’ bands o f P-lg are observed in the deconvoluted FTIR spectrum
o f P-lactoglobulin in the amide I absorption region between 1700 and 1600 cm"1
61
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Figure 4.3). These bands have been previously assigned by other researchers (Susi and
Byler, 1983, Casal et al., 1988, Byler and Farrell, 1989, Ismail et al., 1992, Boye et al.,
1995) as the follows:
the 1692
cm '1 bands is assigned to a high frequency band of
antiparallel P-sheets, hidden in the interior o f the protein, the 1682 cm '1 and the 1675
cm '1 bands are assigned to other high frequency band o f antiparallel P-sheets exposed to
solvent, the 1662 cm '1 band is assigned to p-tums, while the 1645-1649 cm '1 broad band
is assigned to both a-helix and random coil structures. The 1634 cm '1 is assigned to the
low frequency band o f antiparallel P-sheets, while the 1628 and 1624 cm '1 bands are
assigned to extended P-sheets. Boye et al. (1995) also reported that aggregation o f P-lg
also resulted in the appearance o f two additional amide I bands at 1684 and 1618 cm '1
attributed to the formation o f intermolecular antiparallel hydrogen bonded P-sheet.
Antiparallel
P-sheet
3.0
Extended
p-sheet
2.4
2.2 1
a-Helix
a>
o
c
n
si
L_
o
p-Turns
(0
SI
<
Antiparallel
P-sheet
0.4
■j Antiparallel
■I P-sheet
0.2
1700
1680
1660
1640
1620
Wavenumbers (cm'1)
Figure 4.3. The amide I absorption region o f the FTIR spectrum o f [J-lactoglobulin in
D 2 O (5% w/v, p H 6.8), at 25°C after deconvolution. The major bands assigned on each
peak.
62
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4.4.1 Effect o f Elevated Temperatures on the Secondary Structure o f B-lg:
To study the effect o f temperature on the conformation o f |3-lg, variabletemperature FTIR spectroscopy was performed on P-lg in solution Figure 3A. Visual
inspection o f Figure 4.4A shows that the intensity o f the 1692 cm '1 band gradually
decreases with increasing temperature and disappeared at 55°C. This signifies the
complete H-D exchange of the hidden P-structure (Boye et at., 1995). Additional changes
are also observed in the other amide I bands as described below. It is on interest to note
that by incubating p-lg in D 2 O for 50 hr the 1692 cm '1 band is virtually eliminated at
ambient temperatures.(25 °C). Variable-temperature FTIR study o f the H-D exchanged Plg is shown in Figure 4.4B. Marked differences between the two proteins samples can be
discerned.
2 .8
2.6
2.4
2.2
2 .0
0)
o
c
n
-Q
k.
o
(0
n
<
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
- 0.2
1700
1680
1660
1640
1620
Wavenumbers (c m 1)
Figure 4.4A. The deconvolved amide I band in the spectra o f fi-lg (5 % w/v in D 2 O) in the
presence o f the 1692 c m 1 band, upon heating conventionally in the FTIR cell till 55°C
m
63
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
2.8
2.6
2.4
2.2
2.0
a)
u
c
(0
A
L.
o
tn
■q
<
1.8
1 .8
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
1700
1680
1660
1640
1620
Wavenumbers (c m 1)
Figure 4.4B. The deconvolved amide I band in the spectra o f P-lg (5 % w/v in D 2 O) o f HD exchanges fi-lg upon heating conventionally in the FTIR cell till 55°C.
A new band at 1629 cm '1 which has been attributed to P-sheet structure (Boye et
al., 1995) or P-type structure (Krimm and Bandekar, 1986) appeared at 40°C in the
presence of the 1692 cm '1 band, and at 50°C in the absence o f the 1692 cm '1 band,
increased in intensity with increasing temperature. The band at 1682 cm’1 (P-sheet),
started to increase in intensity with increasing temperature. The band at 1676 cm '1 (Psheet) shifted to 1674 cm '1, decreased in intensity with increasing temperature in the
presence of the 1692 cm’1 band. A slight decrease in the band at 1647 cm '1, assigned to ahelical and random coil structures is observed in the P-lg and H-D exchanged p-lg with
increasing temperature. A new band started to appear at -1642 cm ’1 with increasing
temperature, and could be better discerned by plotting the difference spectra in Figures
4.5A and 4.5B (generated by subtracting the ambient temperature spectrum o f P-lg from
all other spectra recorded at higher temperatures). The 1642 cm '1 band is tentatively
assigned to unordered structures. The band at 1632 cm '1 attributed to antiparallel P-sheet
structures decreased in intensity with increasing temperature slowly up to 40°C and more
64
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
rapidly above 45°C. The decrease in the 1632 cm '1 band intensity was greater in (3-lg
compared to the H-D exchanged P-lg. A major decrease in the band intensity at 1622 cm '1
(P-sheet), was observed with increasing temperature, this band disappeared at 55°C in
both P-lg solutions (Figures 4.5A and 4.5B).
It is clear form the above results that H-D exchange is simultaneously occurring
with changes in the secondary structure o f the protein with increasing temperature. In
order to delineate between the two phenomena it is necessary to study the changes in the
secondary structure o f the protein in the fully H-D exchanged P-lg. However, the
sequence o f events leading to changes in the tertiary structure o f the protein can also be
elucidated by studying the changes in the amide I bands sensitive to H-D exchange.
0.09
0.08
i
0.07
a>
o
c
n
0.06 1
0.05
XI
0.04
o
0.03
n
<
0.02
k*
CO
0.01
0.00
- 0.01
- 0.02
-0.03
-0.04
-0.05
-0.06 1
1690
1680
1670
1660
1650
1640
1630
1620
1610
W avenumbers (c m 1)
Figure 4.5A. Superimposed difference FTIR spectra o f /3-lg 5 % (pH 6.8) in D 2 O as a
function o f increasing temperature, after subtraction o f the spectrum recorded at 25°C.
Each line represents spectrum recorded at 5°C increments from 30-55°C.
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
0.09
0.08
0.07
0.06
0.05
0)
o
c
n
JO
io
(/>
XI
<
0.04
0.03
0.02
0.01
- 0.00
- 0.01
- 0.02
-0.03
-0.04
-0.05
-0.06
-0.07
1700
1690
1680
1670
1660
1650
1640
1630
1620
1610
W avenumbers (cm )
Figure 4.5B. Superimposed difference FTIR spectra o f fully H-D exchanged /3-lg 5 % (pH
6.8) in D 2 O as a function o f increasing temperature, after subtraction o f the spectrum
recorded at 25°C. Each line represents spectrum recorded at 5°C increments from 3055°C.
4.4.2 Analysis of the Effect of Temperature on the Amide I’ bands in the Infrared
Spectra of B-lg and H-D Exchanged B-lg by 2D IR Correlation Spectroscopy
The one-dimensional spectral results give an overview o f the unfolding o f p-lg. However,
to discern subtle changes occurring with heating and to deduce the sequence o f unfolding
events, 2D correlation analysis was employed (Ismoyo, et al., 2000). Figures 4.6A and
4.6B show the 2D IR synchronous and asynchronous correlation maps constructed from
the variable-temperature deconvolved FTIR spectra plotted in Figure 4.5A.
66
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Synchronous 2D Correlation Map
1600
1610 1620 VE
1630 - !
|
1640 -
E
ia>
1650 -
I
1660 -
>
1670 1680 1690 -
1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
W avenumber (cm'1)
Figure 4.6A. The synchronous 2D IR spectra generated from the difference spectra
plotted in Figure 4.5A. Positive and negative correlation peaks are represented by solid
and dashed lines, respectively.
67
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Asynchronous 2D Correlation Map
1600
^
1610
|
1620
jj
E
=
1630
1640
O'
a>
«
1650
1660
1670
LJ
1680
1690
1700
1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
W avenum ber (cm'1)
Figure 4.6B. The asynchronous 2D IR spectra generated from the difference spectra
plotted in Figure 4.5A. Positive and negative correlation peaks are represented by solid
and dashed lines, respectively.
68
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Synchronous 2D Correlation Map
160016101620-
□
1630E
1640-
k.
1650-
o
0)
n
E
3
C
<D
>
i
16701680169017001700 1690 1680 1670 1660 1650 1640 16301620 1610 1600
W avenumber (cm'1)
Figure 4.7A. The synchronous 2D IR spectra generated from the difference spectra
plotted in Figure 4.5B. Positive and negative correlation peaks are represented by solid
and dashed lines, respectively.
69
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Asynchronous 2D Correlation Map
16001 6101620-
D
□
1 630E
1 640-
o
■_
<v
n
E
3
C
0)
s
16501 6601 670-
5
16801 69017001700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
Wavenumber (cm'1)
Figure 4.7B. The asynchronous 2D IR spectra generated from the difference spectra
plotted in Figure 4.5B. Positive and negative correlation peaks are represented by solid
and dashed lines, respectively.
Interpretation o f the top half o f the synchronous and asynchronous maps (Figures
4.6A and 4.6B respectively) and with the ID information in the difference spectra in
Figure 4.5A lead the generation o f the sequence o f events shown in table 4.1.
The
procedure generating the sequence o f events for the 2D maps has been described
previously by Wang et al., 1998. Briefly, the first sign in any row or column in the table
is taken from the synchronous map. When this sign is positive (solid line) it means that
the two correlated peaks on X and Y axis, are changing in intensity in the same direction
(either up or down), when the first sign is negative (dashed line), that means the two
correlated peaks are changing in intensity in different directions. W hen the two signs in
any row or column are similar (either two positives or two negatives) it means that the
70
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
changes in the peak on the X axis takes place before the changes on the Y axis. When the
two signs in any row or column are different (either negative-positive or positivenegative), it means that the changes in the peak on the Y axis take place before that on the
X axis. When the arrow is to the right in any row or column it means that the peak on the
X axis change before that on the Y axis, if the arrow is to the left it means that the peak
on the Y axis changes before that on the X axis.
Table 4.1 shows the sequence o f unfolding events upon heating ft-lg till 55 °C.
1692 +
1682 f
1620 |
++
-
1629 |
—
+
1633 |
++
16761
++
16821
—
cm '1
* -
1633 |
1629 f
+
+-
-►
+-
-►
-
-+
-►
-+
-►
++
-►
—
-
*“
16761
*“
- -
<«-
+
The order o f unfolding events o f P-lg is: first the 1692 cm '1 band drops in
intensity, followed by the 1629 cm '1 band, then the band at 1622 cm '1 drops in intensity,
next the 1682 cm '1 then 1633 cm '1 bands drops in intensity and last the 1676 cm '1 band
drops in intensity.
Based on the secondary structure assignment o f the above amide I the mechanism o f heatinduced unfolding o f P-lg before H-D exchange took place involved the initial unfolding
o f the hidden antiparallel P-sheets, followed by an increase in the population o f P-sheets
exposed to solvent. This is followed by a loss o f the extended p-sheets, then a loss on
antiparallel p-sheets and turn structures. The predominate increase in the new P-sheet
structure (1629 cm '1) observed in Figure 4.5A would indicate that the amide I bands
continue to undergo H-D exchange and that the extended P-sheets are transformed to
antiparallel p-sheet structures.
71
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The changes in the band intensity at 1642 cm '1 with increasing temperature were
slight, and overlapped with a-helix band, therefore it was not considered in the sequence
of events, as the two changed in opposite directions with increasing temperature. This
was very apparent when heating the protein solution above 60 °C.
To delineate the changes in the secondary structure 2D correlation were
performed on H-D exchange P-lg. The synchronous and asynchronous maps shown in
Figure 4.7A and 4.7B are shown in table 4.2.
Table 4.2. The sequence o f unfolding events upon heating H-D exchanged f-lg fr o m 25 to
55°C in D20.
cm '1
1682 f
1633 |
1629 f
1622 |
-+
-►
+-
-►
1629 f
+-
—
-+
—
1633 |
--------►
C3
The 1622 cm '1 band drops in intensity first followed by an increase in the intensity
o f the 1629 cm '1 which is then followed a drop in the 1633 cm '1 band intensity which
occurs at the same time as the increase in the 1682 cm '1 band.
Accordingly, after the H-D exchange took place, the unfolding started with an initial
unfolding o f the extended P-sheet followed by the appearance o f solvent exposed p-sheet
structures then the loss o f the antiparallel P-sheet which may also result in an increase in
solvent-exposed P-sheet as evidenced by the continued rise in the 1629 cm '1 band with
temperature in the 1 D spectra (Figure 4.5B). Slight changes were observed in the bands
at 1642 and 1676 cm '1 therefore they were not considered in the sequence unfolding
pathway.
72
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4.4.3 Unfolding of B-Ig after pre-treatment by Microwave or Conventional Heating
at 35°C;
To study the effect o f the microwave irradiation at low temperatures on the
unfolding pathway H-D exchanged P-lg solutions, each solution was subjected to 1-10
cycles o f microwave irradiation to achieve a solution temperature o f 35°C. Each sample
was subjected to VT-FTIR as described above.
Figure 4.8 shows the overlaid FTIR
spectra o f P-lg (irradiated for 3 cycles) in the amide I absorption region. The peaks that
change as a function o f increasing temperature are better defined and more easily
identified in the difference spectra (data not shown).
2.2 1
o
o
c
(0
J3
i_
O
(A
SI
<
0.6
-
0.2 1
-0.4 4
W avenumbers (cm )
Figure 4.8. The overlaid deconvolved o f the VT-FTIR spectra of[3-lg solution exposure to
3 cycles o f microwave irradiation.
Figures 4.9A and 4.9B show the synchronous and asynchronous maps,
respectively, o f P-lg generated from the deconvolved FTIR spectra in Figure 4.8.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Synchronous 2D Correlation Map
160&
161 O'
W avenumber (cm -1)
1 6 2
a '
163a
O
©
C E ila ll
o @
" CD
O
CD
164a
165a
a
166a
1670-
168a
169a
<□
□ CD
.O
170 a
1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
W aven u m b er (cm '1)
Figure 4.9A. The synchronous map generated from a VT-FTIR run (25-55 °C) o f a /3-lg
solution heated 3 times to 35°C by microwave irradiation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Asynchronous 2D Correlation Map
E
o
Si
o
E
3
C
a>
S
£
168a
CD
1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
W avenumber (cm'1)
Figure 4.9B. The asynchronous map generated from a VT-FTIR run (25-55° C) o f a J3-Ig
solution heated 3 times to 35°C by microwave irradiation.
The sequence o f unfolding event can be deduced from the asynchronous maps as
shown in table 4.3.
Table 4.3. The sequence o f unfolding of(3-lg generated from a VT-FTIR run (25-55° C) o f
a (3-lg solution heated 3 times to 35°C by microwave irradiation.
cm"1
1682 f
1622 |
-+
-►
+-
-►
1629 f
+-
—
-+
—
1633 |
1629 f
--
1633 |
75
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The sequence o f unfolding commences with a drop in the intensity o f 1622 cm '1
band followed by an increase in the intensity o f the 1629 cm '1 band which is then
followed by a decrease in the intensity o f 1633 cm '1 along with an increase in the 1682
cm '1 band.
The unfolding pathway o f microwave irradiated P-lg starts with an initial
unfolding o f p-sheet followed by the appearance o f extended P-sheet structures followed
by the drop in the population o f antiparallel P-sheets.
The sequence o f events o f unfolding o f H-D exchanged p-lg solutions irradiated for 4-10
cycles showed similar unfolding pathway as in the 3-cycle microwave irradiation
experiment (data not shown).
The sequence o f events leading to unfolding o f H-D exchanged P-lg exposed to
1-10 heating cycles to reach 35°C in a water bath (conventional heating) followed by
carrying out VT-FTIR spectroscopy studies revealed that a similar unfolding pathway as
in the microwave experiment (data not shown). That means that microwave irradiation or
conventionally heating o f P-lg solution had no effect on the unfolding pathway o f heat
induced unfolding.
4.4.4 2D IR Correlation Analysis of (3-lg Aggregation
To study the effect o f microwave irradiation on the aggregation pathway o f p-lg,
solutions o f P-lg (5 % in D 2 O) were subjected to microwave irradiation till 75°C for
different number o f cycles, 1, 3, 5, 7 and 10 (70 second/cycle) (Figure 4.10A) and
compared with P-lg solutions that had been conventionally heated to the same
temperature 75°C for the same number o f cycles within the same time period (Figure
4.1 OB). Difference spectra generated by subtracting the FTIR spectrum of the 1st
irradiation cycle from the remaining cycles (Figures 4.11 A) were employed to generate
the synchronous and asynchronous maps (Figures 4.12A and 4.12B respectively). The
sequence o f events leading to aggregate formation is shown in Table 4.4. The difference
spectra obtained from conventional heating cycles are shown in Figure 4.1 IB and
employed to generate the synchronous and asynchronous maps (Figures 4.13A and 4.13B
respectively). The sequence o f events leading to aggregate formation is shown in Table
4.5.
76
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0.24
0.22
0.20
Absorbance
0.1 8
0.16
10 cycles
0.1 4
-4 —
0.12
0.10
5 cycles -4
0 .08
0 .06
V
3 cycles 4 ---------- A
0 .04
0.02
0.00
- 0 .02
1700
1680
1660
1640
1620
Wavenumbers (cm'1)
Figure 4.10A. Overlaid deconvolved infrared spectra o f P-lg that subjected to 1,3,5,7,
and 10 cycles o f microwave irradiation till 75°C/cycle.
m
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0.2 4
0.22
0.20
0.18
0.1B
0)
o
c
(0
n
4o
to
-Q
<
0.14
0.12
0.10
0 .06
0 .04
0.02
0.00
- 0.02
1700
1660
1680
1620
1640
Wavenumbers (cm'1)
Figure 4.1 OB. Overlaid deconvolved infrared spectra o f P-lg that subjected to 1,3,5,7,
and 10 cycles o f conventional heating till 75°C/cycle.
t
0.12
0.11
0.10
0.09
0.08
a>
o
c
re
■Q
o
<0
■Q
<
0.07
0.06
0.05
0.04
0.03
0.02
0.01
- 0.00
-0 .0 1
- 0 .0 2
-0.03
-0.04
-0.05
-0.06
-0.07
1700
1690
1680
1670
1660
1650
1640
1630
1620
1610
Wavenumbers (cm'1)
Figure 4.11A . Overlaid FTIR difference spectra o f P-lg 5 % D 2 O as a function o f
irradiation cycles (1,3,5,7 and 10 cycles) till 75°C, after subtraction o f the spectrum
recorded at 30°C.
78
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Wavenumbers (cm'1)
Figure 4.1 l.B . Overlaid FTIR difference spectra o f /3-lg 5 % D 2 O as a function o f
conventional heating cycles (1,3,5,7 and 10 cycles) till 75°C, after subtraction o f the
spectrum recorded at 30°C.
79
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Synchronous 2D Correlation Map
1600 1610 a
1620 -
E
1630 -
<D
JO
1640 -
E
1650 -
3
O
C
a>
%
5
1660 1670 -
o
1680 1690 1700 1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
W avenum ber (cm )
Figure 4.12A The synchronous 2D IR spectra generated fro m the difference spectra in
figure 4.11 A. Positive and negative correlation peaks are represented by solid and
dashed lines, respectively.
m
80
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Asynchronous 2D Correlation Map
16001610-
□
1620-
E
a
1630-
&_
a)
Si
1640-
E
3
C
a>
%
5
1650-
O $
1660167016801690170017001690 168016701660165016401630162016101600
Wavenumber (cm )
Figure 4.12B. The asynchronous 2D IR spectra generated from the difference spectra in
figure 4.11 A. Positive and negative correlation peaks are represented by solid and
dashed lines, respectively.
81
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Synchronous 2D Correlation Map
16001610
[ci) o
1620
I ®
— n (3 ) a
'° !
! .-
1630
a
E
o
1640
■_
o
n
E
0)
3
5
o Q o
....
1650
0
3
C
](§>
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1660
n
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1670
1680
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a
. ( p ; (o>
o ...
□
©
G3) £1)
Qg§j
1690
1700
1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
Wavenumber (cm'1)
Figure 4.13A. The synchronous 2D IR spectra generated fro m the difference spectra in
figure 4.1 IB. Positive and negative correlation peaks are represented by solid and
dashed lines, respectively.
82
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Asynchronous 2D Correlation Map
1600
1610
1620
1630
1640
E
o^
1650
i_
0)
XI
1660
E
3
C
<D
%
5
1670
1680
1690
1700
1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
Wavenumber (cm )
Figure 4.13B. The asynchronous 2D IR spectra generated fro m the difference spectra in
figure 4.1 IB. Positive and negative correlation peaks are represented by solid and
dashed lines, respectively.
83
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Table 4.4 shows the sequence o f unfolding events o f f- l g after subjected to microwave
irradiation cycles to achieve 75 °C.
1682 f
16761
1615 |
+-
..
<—
- -
1620 |
-+
+-
—►
+-
—►
1633 |
-+
++
^__
++
<__
1640 |
+-
-+
16471
-+
. . <«—
1676 |
-+
cm '1
—►
*
1647 |
4—
1640 f
1632 |
1620 |
+ + <—
-+
. .
-- *
+-
"
-►
-+
—►
4-
^
* -
-►
The sequence of events commences with an increase in the random coil followed
by a decrease in the P-sheet structures which is followed by the decrease in the a-helix,
and finally the formation o f intermolecular antiparallel p-sheet.
A similar unfolding
sequence was observed when P-lg was conventionally heated for the same number of
cycles, to the same temperature 75°C within the same time frame. It should be noted that
the amount of aggregation was significantly higher in the microwave irradiated sample
(Figure 4.14). Thus, the microwave irradiation does not alter the unfolding pathway but
catalyzes the unfolding process.
84
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0.24
0.22
0.20
®
o
c
n
.Q
o
0.18
0.16
0.12
(0
0.10
<
0.08
.Q
10 Cycles
MW
0.14
0.06
10 Cycles
C.V.H
0.04
0.02
- 0.00
- 0.02
1700
1680
1660
1640
1620
W avenum bers (cm'1)
Figure 4.14. Comparison between the effect o f 10 microwave irradiation and
conventional heating cycles till 75°C on the extent o f aggregation o ff-lg .
4.5 Discussion and Conclusion:
X-ray crystallography o f bovine p-lactoglobulin revealed that P-lg consists o f
51.2% p -sheets and 32.1% random coil, and 9.9% P-tum, and 6.8% a-helix (Qi, et al.,
1997). Employing CD spectroscopy (Qi, et al., 1997) showed that P-lg in solution
consists o f 50 % P-sheet, random coil 35% , P-tum 8 % , and helix 10 %. Thermal
denaturation o f P-lg involves the dissociation o f bovine p-lg dimer, unfolding and
aggregation (Burova, et al, 2002). A variable temperature study by Boye et. al, 1995,
employing FTIR spectroscopy of B-lg in phosphate buffer, pH 7, at temperatures between
30°C and 95°C suggested that the mechanism o f heat denaturation o f P-lg involves an
initial unfolding o f a P-type structure in the interior o f the protein, which results in the
formation of more extensive random coil stmctures, followed by an unfolding o f the ahelical structure and formation o f intermolecular p-sheets, resulting in aggregate
formation, This is consistent with the results we obtained up on heating P-lg solution till
55°C, however, the changes in the a-helix stmctures were minor when the protein was
heated to 55°C. The order o f unfolding event before aggregation upon heating P-lg started
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with the unfolding o f P-sheet, this is in agreement with Boye et al, 1995. Qi et al., 1997
using CD and FTIR showed that p-lg progressively lost p-sheet structure with increasing
temperature, and lost almost all the helical structures at 65°C. Above 65°C a partly
unfolded state is formed, possibly by destabilization o f the intramolecular P-strand I and
the loss o f the main helix. These researchers found that at physiological concentrations
the dimer disassociates coupled with a conformational transition to an R-type state
between 40 and 55°C. The R-state differs from the native conformation in the solution by
increased accessibility o f a free thiol group to intermolecular reactions and is not
accompanied by major changes in the protein secondary structure.
The R-state may be attributed to the relaxation o f the protein tertiary structure
allowing solvent to enter the interior o f the protein (resulting in the H-D exchange
evidenced by the decrease in the 1692 cm '1 band). The exposure o f the free thiol may also
be accompanied by the increase in unordered structure (the appearance o f a new band at
1642 cm '1). The sulfhydryl group o f Cysl21 may contribute to the maintenance o f P-lg
tertiary structure via water mediated H-bonding (Burova et al., 1998). A role of the Cys
121 should be underlined when thermal unfolding o f p-lg is discussed. Cys 121 is buried
at the interface between p-sheet and a-helix. It was suggested that irreversibility o f P-lg
denaturation results mainly from the formation o f intermolecular and nonnative
intramolecular disulfide bonds initiated by a single sulfhydryl group during unfolding of
P-lg (McKenzie and Sawyer, 1967; McKenzie and Ralston, 1973).
The dissociation o f the dimer to monomer results in a decrease in an antiparallel
P-sheet (Casal, 1988). In this work the increase in the 1629 cm '1 band is assigned to an
increase in extended p-sheet which is formed from the dissociation o f an antiparallel Psheet holding the two P-lg monomers. This is in good agreement with x-ray data (Casal
et al., 1988) from which suggested that an exposed P-strand forms an intermolecular
antiparallel P-sheet upon dimer formation (Casal et al., 1988). Irreversible conformational
changes o f B-lg occur at a temperatures above 65 °C, at this temperature B-lg, aggregates
due to spontaneous interaction arising from partial unfolding o f the molecule, which
releases previously committed hydrogen bonded protein groups for alternative interaction,
although the specific structure o f aggregates is unknown (Sawyer et al., 1971).
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Nonthermal effect o f microwave irradiation have been reported to affect the
kinetics o f protein folding processes o f a number o f globular proteins particularly (3-lg
(Bohr, et al., 1999, 2000). From the results o f the above work, we found that the
microwave irradiation produced a higher p-lg aggregation than by conventional heating
processes. In other words the nonthermal effects o f microwave irradiation were more
efficient in producing intermolecular hydrogen bonded P-sheet. 2D correlation analysis
revealed that the unfolding sequence o f P-lg was similar. Accordingly, the nonthermal
effect o f the microwave accelerated (i.e., catalyzed) the process o f unfolding rather than
targeting specific domains within the protein leading to an altered unfolding pathway.
The nonthermal effect appears to be cumulative as increasing the number of
cycles resulted in a higher degree o f H-D exchange at lower temperatures and
significantly higher degree o f aggregation at higher temperature approaching the Tm o f plg. This suggests that the protein structure retains the nonthermal effect “memory effect”.
Accordingly, the nonthermal effect maybe attributed to a drop in the activation energy of
the protein unfolding. Future studies will be directed at the determination o f the impact of
the nonthermal effect on the AG, AH and AS o f P-lg.
87
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REFERENCES
1- Bohr, H. and Bohr, J., 2000. Microwave-enhanced Folding and Denaturation o f
Globular proteins, The American Physical Society, V 61, number 4.
2- Bohr, H. and Bohr, J., 2000, Microwave Enhanced Kinetics observed in ORD
Studies o f a Protein. Bioelectromagnetics 21:68-72.
3- Bohr, H., Bohr, J., Brunak, S., 1997, Protein Folding and Wring Resonances.
Biophysical Chemistry 63, 97-105.
4- Boye, J. I.; Ma, C.-Y.; Ismail, A. & Alii, I. J. 1996. Effect o f Physico-chemical
Factors on the Secondary Structure o f P-lactoglobulin Journal Dairy Research , 63 ,
97-109.
5- Burova, T.V., Grinberg, N.V., Visschers, R.W., Grinberg, V.Y., and de Kruif, G.C.
2002. Thermodynamic Stability o f Porcine P-lactoglobulin. A structural Relevance
Eur. J. Biochem. 269, 3958-3968.
6- Burova, T.V., Choisetl, Y, Tran, V., and Haertle, T., 1998. Role o f free Cysl21 in
Stabilization of Bovine p-lactoglobulin B. Protein Engineering vol. 11 no. 11
pp. 1065-1073.
7- Casal, H.L., Kohler, U., Mantsch, H.H. 1988. Structural and conformational
changes o f P-lactoglobulin B: and infrared spectroscopic study o f the effect o f pH
and temperature. Biochim. Biophys. Acta, 957, 11.
8- Chikenji, G., and Kikuchi, M, 2000. W hat is the Role o f Nonnative intermediates o f
P-lactoglobulin in Protein Folding? Proc Natl A cad Sci U S A. 19; 97 (26): 1427314277.
9- D ’Alfonso, L., Collini, M. and Baldini, G., 2002. Does P-lactoglobulin Denaturation
Occur via an Intermediate State? Biochemistry 41, 326-333.
10-De Pomerai, D.I., Smith, B., Dawe, A., North, K., Smith, T., Archer, D.B., Duce,
I.R., Jones, D., Peter, E., Candido, M., 2003. Microwave Radiation can Alter
Protein Conformation without Bulk Heating. FEBS Letters 543, 93-97 Published by
Elsevier Science B. V. May 2003.
8- Englander, J.J., Mar, C.D., Li, W., Englander, S.W., Kim, J.S., Stranz, D.D.,
Hamuro, Y. and Woods V.L., 2003. Protein Structure Change Studied by Hydrogen-
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deuterium Exchange, Functional Labeling,
and mass
Spectrometry. PNAS,
june 10,2003, Vol.100, no. 12, P 7057-7062.
9- Englander, S.W., and Krishna, M.M.G., 2001. Hydrogen Exchange
Nature Structural Biology 8 1-2.
10-Forge, V., Hoshino, M., Kuwata, K., Arai, M., Kuwajima, K., Batt, A.C. and Goto,
Y., 2000. Is Folding o f P-lactoglobulin Non-hierarchic? Intermediate with Native­
like P-Sheet and Non-native a-Helix. J.Mol.Biol. 296, 1039-1051.
11-Hoedemaeker, F.J; Visschers, R.W; Alting, A.C; de Kruif, K.G; Kuil, M.E and
Abrahams, J.P, 2002. A novel pH-dependent dimerization m otif in P-lactoglobulin
from
pig
(Sus
scrofa).
Acta
Crystallographica,
Section
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Biological
Crystallography D58, 480-486.
12-Ipsita, R.; and Munishwar, N.G., 2003. Non-thermal Effects o f Microwaves on
Protease-catalyzed Esterification and Transesterification. Tetrahedron 59, 54315436.
13-Ismoyo, F., Wang, Y., and Ismail, A. A., 2000. Examination o f the Effect of Heating
on the Secondary Structure o f Avidin and Avidin-Biotin Complex by ResolutionEnhanced 2D Infrared Correlation Spectroscopy. Appl. Spectrosc. 54:939-947.
14-Kuwata, K., Hoshino, M., Era, S., Batt, A.C. and Goto, Y. 1998. a - > P Transition
o f p-Lactoglobulin as Evidenced by Heteronuclear N M R J. Mol. Biol. 283(4), 731 739.
15-Kuwata, K., Shastry, R., Cheng, H., Hoshino, M., Batt, C.A., Goto, Y., and Roder,
H., 2001. Structureal and Kinetic Characterization o f early folding events in plactoglobulin. Natural structural biology. V.8.number 2. 2001.
16-Kaufmann, B and Christen, P 2002. Recent Extraction Techniques for Natural
Products: Microwave-assisted Extraction and Pressurized Solvent Extraction
Phytochemical Analysis. V.13, 105-113.
17-Koutchma, T., LeBail, A. and Ramaswamy, H.S, 2001. Comparative Experimental
Evaluation
of Microbial
Destruction
in
Continuous-flow
Microwave
and
Conventional Heating Systems. Canadian Biosystems Engineering 43, 3.1-3.8.
18-La Cara, F., Scarfi, M.R., D ’Auria, S., Massa, R., D ’Ambrosio, G., Franceschetti,
G., Rossi, M. and De Rosa, M., 1999. Different Effects o f Microwave Energy and
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conventional heat on the activity o f a thermophilic P-galactosidase from Bacillus
acidocaldarius. Bioelectromagnetics 20:172-176.
19-Laurence, J.A.; French, P.W.; Lindner, R.A. and McKenzie, D.R., 2000. Biological
Effects o f Electromagnetic Fields-Mechanisms for the Effects o f Pulsed Microwave
Radiation on Protein conformation. J.Theor. Biol. 206, 291-298.
2 0 -Monahan, F.J., German, B. and Kinsella, E., 1995, Effect o f pH and Temperature on
Protein Unfolding and Thiol/Disulfide Interchange Reactions during Heat-Induced
Gelation o f Whey Proteins. J. Agric. Food Chem. 43, 46-52.
21-M udgett, R.E., 1986. Microwave Properties and Heating Characteristics o f Foods
Food Technol. 40, 84-93.
2 2 -Peinnequin, A., Piriou, A., Mathieu, J., Dabouis, V., Sebbah, C., Malabiau, R.,
Debouzy, J.C., 2000. Nonthermal Effects o f Continuous 2.45 GHz Microwaves on
Fas-induced Apoptosis in Human Jurkat T-cell Line. Bioelectrochemistry and
Bioenergetics. 51:157-161.
23-Porcelli, M; Cacciapuoti, G; Fusco, S; Massa, R; d ’Ambrosio, G; Bertoldo, C; De
Rosa, M; Zappia, V. 1997. Nonthermal effects o f microwaves on proteins:
thermophilic enzymes as model system. FEBS Lett. 402(2-3): 102-6.
24-Ptak, D, 1998. Rigid-body oscillations o f a-helices: implications for protein thermal
stability. Biophysical Chemistry 73 121-127.
2 5 -Qi, X., Holt, C., Mcnulty, D., Clarke, D.T., Brownlow, S. and Jones, R.G., 1997.
Effect of Temperature on The Secondary Structure o f P-lactoglobulin at pH 6.7, as
Determined by CD and IR Spectroscopy: a Test o f The Molten Globule Hypothesis
Biochem.J. 324, 341-346.
26- Sato, S., Shibata, C. and Yazu, M., 1996. Non-thermal Killing Effect o f Microwave
Irradiation. Biotech. Tech., 10, 145-150.
2 7 -Smith, L., Deng, Y. and Zhang, Z., 1997. Probing the Non-covalent Structure o f
Proteins by Amide Hydrogen Exchange and Mass Spectrometry. Journal o f Mass
Spectrometry, 32, 135-146.
28- Steel, B.C., Bilek, M.M., McKenzie, D.R., dos Remedios, C.G, 2002. A technique
for microsecond heating and cooling o f a thin biological sample. Eur Biophys J 31
378-382.
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29-Taulier, N and Chalikian, T.V, 2001. Characterization o f pH-induced Transitions of
P-lactoglobulin: Ulterasonic, Densimetric, and Spectroscopic Studies. J.Mol.Biology
314, 873-889.
30-Taylor, L.S., 1981. The Mechanisms o f Athermal Microwave Biological Effects.
Bioelectromagnetics 2, 259-267.
31-Ugolini, R; Ragona, L; Silletti, E; Fogolari, F; Visschers, R.W; Alting, A.C and
Molinari, H, 2001. Dimeriaztion, stability and electrostatic properties o f porcine Plactoglobulin J.Biochemistry 268, 4477-4488.
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CHAPTER 5
Nonthermal Effects o f Microwave Irradiation
on Hemoglobin
5.1 ABSTRACT
This study involves the application o f FTIR spectroscopy, circular dichroism and
fluorescence spectroscopy techniques to investigate the nonthermal effects o f microwave
irradiation on hemoglobin conformation. Irradiation and conventional heating at
temperatures between 25 and 48 °C, revealed no differences between the two treatments,
but at 54°C especially after 4 heating cycles the difference was highly significant.
Hemoglobin exposed to microwave irradiation aggregated faster than thermal treatment,
in addition, when the number o f cycle increased, the microwave effect was progressively
amplified. Fluorescence spectroscopy revealed that the nonthermal effect resulted in the
tyrosine and tryptophan residues becoming more solvent. There was also a red shift in the
wavelength in the irradiated samples compared to the thermal treatment. At 54°C the
amount o f protein aggregation was substantially higher than the thermally treated samples
as evidenced by the higher intensity o f 1684 and 1616 cm '1 in the amide I absorption
region in the FTIR spectra o f hemoglobin in D 2 O. The results indicate that the nonthermal
effects decrease the activation energy o f the unfolding process catalyzing the formation o f
intermolecular p-sheet.
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5.2 INTRODUCTION
In recent years there has been considerable interest in the development of
microwave-based food processing technologies. Microwaves possess electric and
magnetic fields, which are perpendicular to each other. The electric field causes heating
via two simultaneous mechanisms, namely, dipolar rotation and ionic conduction
(Thuery, 1992; Demesmay and Olle, 1993, Sinquin et al., 1993), also microwaves occupy
the segment o f the electromagnetic spectrum between 300 MHz and 30 GHz; however,
microwave-heating applications are commonly limited to a discrete set o f frequencies
reserved for industrial, scientific, and medical use (Dibben, 2001).
Studies o f the effects o f microwave on bacteria, viruses and DNA were
implemented in the 1960s and comprised research on heating, biocidal effects, dielectric
dispersion, mutagenic effects and induced sonic resonance. The majority of the biologists
declared there was no indication of a microwave effect (Goldblith 1967, Lechowich 1969,
Vela 1978, Jeng 1987, Fujikawa 1991, W elt 1994). Some o f the biophysicists investigated
the microwave absorption provided evidence o f a microwave effect which was discrete in
its biocidal effects from the effects o f external heating (Cope 1976, Barnes 1977, Furia
1986).
The thermal effects are related to the heat generated by the absorption of
microwave energy by water or by organic molecules, but very little is known about the
molecular mechanisms engaged in the presumed nonthermal effects. It has been
postulated that the latter might involve a direct energy transfer from the electromagnetic
field to the vibrational modes o f the macromolecules, changing their conformation
(Porcelli et al., 1997). Recently, Bohr et al. demonstrated that microwave irradiation can
affect the kinetics o f the folding process o f some globular proteins (Bohr et al., 1997,
2000 ).
Hemoglobin was chosen as a model protein in this study because it is a tetrameric
protein with a well characterized secondary and tertiary structure. It consists o f 75 %
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alpha helices and has 6 tryptophans, 12 tyrosines, and 30 phenylalanines making it ideal
for study by fluorescence and CD spectroscopy.
Heme
Heme
Heme
Heme
Heme
Figure 5.1 Hemoglobin with the subunits displayed in ribbon representation (The
coordinates fo r the hemoglobin protein were determined using x-ray crystallography, and
the image was rendered using SwissPDB Viewer and POV-Ray).
5.3 Materials and Methods
Human hemoglobin was purchased from Sigma (St. Louis, MO).
D 2 O (99.9
atom % D) was purchased from Aldrich (Milwaukee, WI), and used without additional
purifications.
50 mg/ml (5 % w/v) solutions were prepared in D 2 O, H 2 O, (pH~7), and in 0.2M
phosphate buffer (pH 7) separately. All samples for the FTIR, CD and fluorescence
studies were prepared in triplicates.
5.3.1 Microwave Treatments:
Samples were subjected to Synthewave402 microwave (PROLABO, 54, rue
Roger Salengro - BP 115 - 94126 FONTENAY-SOUS-BOIS CEDEX). This system uses
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a focused microwave and operates at a frequency o f 2.45 GHz (X, = 12 cm), with an
adjustable power between 15 and 300 Watts.
Hemoglobin (Hb) samples were prepared from bulk solution in triplicates, (1 ml
o f Hb solution 5% in each tube), and placed in the cavity o f the microwave. Each sample
was and subjected to eight different temperatures: 30, 35, 40, 44, 48, 50, 54 and 55°C.
Microwave cycles (pulses) o f 1-4 were used. In each cycle treatment, samples were
subjected to microwave irradiation till they reached the desire temperature, then
microwave were turned off, samples were taken out, left un till they reached room
temperature 25°C prior to re-heating to the target temperature. All the spectroscopic
measurements were performed at 25°C. Irradiation time varied from 15 to 50 seconds
depending on the target temperature.
5.3.2 Conventional Heating Treatments:
Hemoglobin samples were prepared from bulk solution in triplicates, (1 ml P-lg 5
% w/v solution in each tube), samples were placed in a water bath for 1-4 cycles till they
reached the desired temperature. The heating and holding time were exactly the same
employed in the microwave experiment, this was achieved that by adjusting the
temperature o f the water bath.
5.3.3 Fourier Transform Infrared (FTIR) Spectroscopy:
FTIR spectroscopy samples were recorded on a M agna 55 FTIR spectrometer
(Nicolet, Madison, WI) equipped with a DTGS detector. The spectrometer was purged
with dry air by a Balston dryer (Balston, Haverhill, MA, 01835-0723, USA).
Approximately 8 pi o f a 5 %( w/v) Hb solution was placed between two CaF 2
windows separated by a 50 pm spacer. A total o f 512 scans were co-added at 4 cm '1
resolution. The temperature o f the sample was regulated by placing the cell in a
temperature-controlled holder employing an Omega temperature controller. The reported
temperatures are accurate to within ± 0.5 °C.
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Mathematical resolution enhancement of the infrared spectra was performed using
Fourier transform deconvolution algorithm as described by Kauppinen et al., 1981. A half
bandwidth o f 18.4cm'1 and enhancement factor (k) o f 2.4 was used on all spectra. All
Spectra were normalized by measuring the peak heights o f the main amide I bands, aHelix (1653 cm '1), 3io-Helix (1636 cm '1), 13-sheet (1617 and 1683 cm '1), and dividing the
peak heights by the peak area o f the amid I’ absorption region (1700-16000 cm’1).
5.3.4 Fluorescence Spectroscopy;
Hemoglobin solutions (0.1 mg/ml) were prepared by a 500-fold dilution o f the 50
mg/ml solutions (in H 2 O, or 0.2 M phosphate buffer) that had been subjected to
microwave irradiation, or conventional heating treatments.
500-pi samples were placed in 1cm quartz cuvette and scanned using an Aminco
Bowman series II fluorophotometer (Rochester, NY) in the wavelength range between
280 and 400 nm. Excitation parameters: step size: 1 nm, wave length: 295 nm, or 285 nm,
band pass: 8 nm. Emission parameters: step size: 1 nm, wave length: 320 nm, band
passes: 4 nm. Emission scan: 280-400nm, scan rate 1.00 nm/sec
5.3.5 Circular Dichroism;
CD spectra were recorded using J-710 spectropolarimeter (Japan Spectroscopic,
Tokyo, Japan), operated by Jasco software. Hemoglobin solutions (0.1 mg/ml) were
prepared by a 500-fold dilution o f the 50 mg/ml solutions (in H 2 O, or 0.2 M phosphate
buffer) that had been subjected to microwave irradiation, or conventional heating
treatments.
5.3.5.1 Far UV CD:
300-pl samples were placed in a 0.1cm quartz cuvette and scanned in the
wavelength range between 190 and 260 nm at a resolution o f 0.2 nm with a scan rate o f
50 nm/min, a
bandwidth of 1 nm, response: 0.25 second, sensitivity: 50, and 5
accumulations. The spectra o f the water or the buffer, were subtracted from each sample
spectra, noise reduction was used to smooth the spectra.
96
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5.4. Results and Discussion:
5.4.1 Fourier Transform Infrared Spectroscopy
Figure 5.2 shows the Fourier self deconvolved FTIR spectrum o f Hb in D 2 O in
amide I absorption region (1700-1600) cm '1. Two strong bands at 1653 cm '1 (assigned to
a-H elix and at 1636 cm '1 (assigned to 3 1 0 -Helix) are observed. A week shoulder at 1675
cm '1 is attributes to turns.
CO
m
co
CO
O
oJ
0
to
O
2
<
0.6
0.4
0.2
0.0
1700
1680
1660
1640
1620
W a v e n u m b e r s (cm-1)
Figure 5.2. FSD spectrum o f Human hemoglobin in D 2 O at 25°C in the amide / ’
absorption region.
FTIR spectra of hemoglobin conventionally heated and microwave treated to 35°C
and 48°C for both two and four cycles were compared. No significant differences in the
a-helical content between the two treatments were observed at these temperatures (data
not shown). Heating Hb solution to 54°C resulting in the appearance o f two additional
97
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amide I bands at 1683 and 1617 cm '1assigned to intermolecular (3-sheet formed from the
unfolding o f the native structure (Figures 5.3 and 5.4). The results show that at 54°C the
amount o f intermolecular P-sheet formed was greater in the microwave treated samples
than in the conventionally heated samples. Figures 5.5 and 5.6 show the plots o f the a helical content and intermolecular P-sheet formation as a function o f heat treatment at
different temperatures.
<u
0.9
eo
0.8
<
0 .7 1
0.6
0 .5 1
MW
0.3
0 .2
0.1
1700
1680
1660
1640
1620
W a v e n u m b e rs (cm -1)
Figure 5.3. FSD-FTIR spectrum o f Hb in the amide I absorption region o f Hb heated
twice to 54 C° by microwave irradiation (MW) and conventional heating treatment.
98
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1700
1680
1660
1640
1620
W a v en u m b ers (cm -1)
Figure 5.4. FSD-FTIR spectrum o f Hb in the amide I absorption region o f Hb heatedfour
times to 54 C ° by microwave irradiation (MW) and conventional heating treatment.
The a-helical content in the microwave treated samples (at 54°C) was lower
compared to conventionally heated samples under the same conditions. This effect was
amplified with increasing number of heating cycles, illustrating that the nonthermal effect
o f microwave treatment is additive. Thus, the irradiate protein appears to have a
“memory” effect albeit the remaining secondary structure appears to be same as the
conventionally heated Hb.
99
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Temperature(C)
Figure 5.5. A plot o f the peak height o f the 1683 cm'1 band assigned to intermolecular (5sheets in the infrared spectra o f hemoglobin heated fo r 4 cycles by microwave irradiation
(black) or conventional heating (white) at 35, 48 and 54°C.
T e m p e ra tu re (C)
Figure 5.6.
A p lo t o f the peak height o f the 1653 c m 1 band assigned to a-helical
structures in the infrared spectra o f hemoglobin heated fo r 4 cycles by microwave
irradiation (black) or conventional heating (white) at 35, 48 and 54°C.
100
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It is of interest to note that the use of microwaves to warm blood products came
into use in the 1970s. But the development of hemolysis as a result o f general overheating
and local hot spots within the blood units was an important concern w ith the use o f these
microwave wormers. It is reported that localized overheating o f packed red blood cells
after microwave worming with consequent damage to erythrocytes (Hirsch, et al., 2003).
5.4.2 Fluorescence:
Fluorescence spectra were acquired from diluted samples o f hemoglobin, heated
conventionally and by microwave irradiation. Figure 5.7 shows the fluorescence spectra
of the native hemoglobin at 25°C, that was excited at 280 nm, with A.max at 331 nm,
attributed to the tryptophan residues, and a weak shoulder at ^ max 303 nm, that is assigned
to tyrosine residues (weak due to energy transfer from tyrosine to tryptophan).
10
8
a>
o
c
a>
o
V)
a>
6
4
2
0
290
305
320
335
350
365
380
395
Emission
Figure 5.7. Fluorescence emission spectrum o f native hemoglobin at 25 °C, excited at 280
nm
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According to the literature, the A ^x o f tryptophan fluorescence spectra shifts to
longer wavelength (red shift) and the intensity of A^ax decrease as the polarity o f the
solvent increase (Weber, G., 1960; Burstein et al., 1973). Maximum values o f
Amax
of
tryptophan have been observed over a broad range between 308 nm (highly non-polar),
and 352 nm (fully exposed to solvent) (Pain.H.R, 1996). On the other hand, the
wavelength o f tyrosine fluorescence does not vary with the polarity o f environment.
Phenylalanine does not absorb above 275 nm so that its weak fluorescence is not
normally observed.
The fluorescence spectrum o f hemoglobin at 35°C in both microwave and
conventionally heated samples, shows no shifting in A ^x o f tryptophan in both
treatments, but a slight difference in
Amax intensity, which might be
due to a difference in
concentration.
The fluorescence spectrum o f hemoglobin at 48°C, after four heating cycles (Figure 5.8),
and at 54°C after two heating cycles (Figure 5.9) show Amax o f tryptophan in the
microwave treated samples, are red shifted (from 331 nm to 333 nm at 48°C and from 331
nm to 338 nm at 54°C), compared to conventionally heated samples (no shift in
Amax at
48°C and from 331 nm to 333 nm at 54°C). These results indicate that hemoglobin in the
microwave treated samples are more solvent exposed compared to the conventional
heating method.
The extent o f unfolding is both irreversible and cumulative and is
sensitive to both the irradiation temperature and the number o f irradiation/heating cycles.
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10
8
6
4
2
0
290
310
330
350
370
390
Emission
Figure 5.8. Fluorescence spectra o f hemoglobin at 48°C using an excitation at 280 nm
after fo u r o f microwave irradiation cycles (dashed line), or after fo u r conventional
heating cycles (solid line).
10
8
6
4
2
0
305
325
345
365
385
E m ission
Figure 5.9. Fluorescence spectra o f hemoglobin at 54°C using an excitation at 280 nm
after four o f microwave irradiation cycles (dashed line), or after fo u r conventional
heating cycles (solid line).
103
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W hen Hb samples were excited at 295 nm, similar fluorescence results were
obtained (as discussed previously for the excitation at 280 nm), with less difference
between the microwave treated and the conventionally heated methods. ^ max of
microwave treated samples red shifted (from 336 nm, to 337 nm at 48°C, four cycles, and
from 336 nm, to 339 nm, at 54°C, for two and four heating cycles). A smaller red-shift
was observed in the conventionally heated samples (no shift at 48°C, four cycles, and a
small red shift from 336 nm to 337 nm, at 54°C, for both the two and four heating cycles).
Two kinds o f water molecules are associated with proteins: internal water that fill
the cavities within proteins, and a peripheral hydration shell on the exterior o f proteins
(Saenger et al., 1987). Microwave may affect the structure o f water at the protein/water
interface and/or within the protein directly or indirectly, which may induce some changes
in both the interior hydrophobic environment and the surface hydrophilic environment o f
this protein. This might explain the difference between the microwave irradiation and the
conventional heating treatment on hemoglobin solutions in the results we obtained.
5.4.3 C ircu lar D ichroism CD:
The far-UV or amide region (170-250) nm, is dominated by contributions o f the
peptide bond, whereas CD bands in the near-UV region (250-300) nm originate from the
aromatic amino acids. In addition, disulphide bonds give rise to minor CD bands around
250 nm. Each spectral region gives different types o f information about the protein
structure. CD bands in the amide region contain information about the peptide bonds and
the secondary structure o f a protein and are frequently employed to monitor changes in
secondary structure in the course o f structural transitions (Schmid, 1989).
Native hemoglobin gives the characteristic spectra o f a mostly a-helix protein with
minima at 208 and 222 nm (Figure 5.10).
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70
CD
-40
190
W avelength[nm ]
260
Figure 5.10. Far UV CD spectrum hemoglobin at 25°C.
Our results revealed that both microwave treated and conventional heated samples
have very similar characteristic shape. However hemoglobin lost part o f its secondary
structure (a-helix), gradually with increasing temperature in both treatments. Slight
differences in the secondary structure (intensity and shape o f spectra) between the
microwave-treated and conventional heated samples were observed with increasing
temperature (Figure 5.11). The microwave irradiated Hb samples lost higher amount of its
secondary structure compared to the conventionally heated samples. These results are
consistent with the results obtained from FTIR spectroscopy and fluorescence studies.
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60
CD
-40
1 95
W avelength[nm ]
260
Figure 5.11. Far UV CD spectrum hemoglobin at 54°C after fo u r heating cycles, by
microwave irradiation (dashed line), and conventional heating (solid line)
5.5 CONCLUSION
Conformational changes in the tertiary and secondary structure of hemoglobin
occur with increasing temperature. Fluorescence studies also confirmed the existence o f a
nonthermal effect o f microwave irradiation, the interior o f the protein was more solvent
exposed and the solvent accessibility increased with the number o f irradiation cycles.
These changes were more significant at higher temperatures. FTIR studies revealed that
intermolecular p-sheet formation was significantly higher in microwave irradiated
samples. This increase is attributed to nonthermal effects o f microwave irradiation which
leads to a lowering o f the activation energy o f the unfolding process. The nonthermal
effect is cumulative and appears to be irreversible within the time frame o f the irradiation
experiments. The results o f the Far UV CD were also consistent with the FTIR
spectroscopic analysis and the fluorescence results.
106
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REFERENCES
1. Alben, J.O and Bare, G.H., 1980. Ligand-dependent Heme-protein Interaction in
Human Hemoglobin Studied by Fourier Transform Infrared Spectroscopy. The
journal o f biological chemistry 255, 3892-3897.
2. Bohr, H. and Bohr, J., 2000. Microwave Enhanced Kinetics Observed in ORD
Studies o f a Protein. Bioelectromagnetics 21:68-72.
3. Datta, A.k., and Anantheswaran, R.C, 2001. Handbook o f Microwave Technology
for Food Applications, p: 1, 191-199.
4. De Pomerai, D.I., Smith, B., Dawe, A., North, K., Smith, T., Archer, D.B., Duce,
I.R., Jones, D., Peter, E., Candido, M., 2003. Microwave Radiation can Alter
Protein Conformation without Bulk Heating. FEBS Letters 543, 93-97 Published
by Elsevier Science B. V. May 2003.
5. Determining the CD Spectrum of a protein, CHEM 690 course, Concordia
University, course note, 2003.
6. Determining the Fluorescence Spectrum o f a protein, CH EM 690 course,
Concordia University, course note, 2003.
7. Geraci, G. and parkhurst, L. 1981. Circular Dicroism Spectra o f Hemoglobin
Methods in enzymology. 76, 292-275.
8. Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB
Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.
9. Hirsch, J., Menzebach, A., Welters, D.I., Dietrich, V.G., Katz, N., and
Hempelmann, G. 2003. Indicators o f Erythrocyte Damage after Microwave
Warming o f Packed Red Blood cells. Clinical chemistry 49:5 792-799.
10. James, C. W., Chien and Fred W., Snyder, JR. 1976, Allosteric Transitions in
Cobalt Hemoglobins. The journal o f Biological Chemistry, Vol. 251. No. 6. pp.
1670-1674.
11. Kiel, J.L., Erwin, D.N., 1986. Microwave Radiation Effects on the Thermally
Driven Oxidase o f Erythrocytes. Official Journal o f European Society fo r
Hyperthermia Oncoloy. 2(2), 201-212.
107
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12. Kiel, J.L., McQueen, C., Erwin D.N., 1988, Green Hemoprotein of Erythrocytes:
Methemoglobin Superoxide Transferase. Physiological Chemistry and Physics
and Medical NM R 20(2), 123-128.
13. Liu, C., Bo, A., Cheng, G., Lin, X., Dong, S, 1998. Characterization o f The
Structureal and Functional Changes o f Hemoglobin in Dimethyl Sulfoxide by
Spectroscopic Techniques. Biochimica et Biophysica Acta 1385, 53-60.
14. Li, R., Nagai, Y. and Nagai, M. 2000, Changes o f Tyrosine and Tryptophan
Residues in Human Hemoglobin by Oxygen Binding: near-and far UV circular
dichroism o f isolated chains and recombined hemoglobin. Journal o f inorganic
biochemistry. 82, 93-101.
15. Perutz, M.G., 1964. The Hemoglobin Molecule, printed from Scientific American
November 1964, P: 3, 4.
16. Porcelli, M; Cacciapuoti, G; Fusco, S; Massa, R; d ’Ambrosio, G; Bertoldo, C; De
Rosa, M; Zappia, V. 1997. Nonthermal effects o f microwaves on proteins:
thermophilic enzymes as model system. FEBS Lett. 402(2-3): 102-6.
17. Casiday, R., and Frey, R. Hemoglobin and the Heme Group: M etal Complexes in
the Blood for Oxygen Transport, Inorganic Synthesis Experiment Department of
Chemistry, Washington University, course tutorial. Internet website:
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComple
xinBlood.html.
18. Schmid, F.X. 1989. Spectral Methods o f Characterizing Protein Conformation and
Conformational Changes. In Protein Structure (Creighton, T. E., ed.), pp. 251
275. IRL Press, Oxford.
108
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CHAPTER 6
General Conclusion
FTIR spectroscopy, CD and fluorescence techniques were employed in this thesis
to reveal that nonthermal effects cause conformational changes in the secondary and
tertiary structure of proteins.
The effect o f multiple-cycle microwave irradiation at 2450 MHz at 35°C on the
extent of H-D exchange of the amide groups revealed that the nonthermal effect was
responsible for dramatically altering the tertiary structure o f the (f-lg allowing solvent to
enter the interior o f the protein.
The nonthermal effect was sensitive to the pH o f the solution. The nonthermal
effect o f microwave is also dependent on the overall charge on P-lactoglobulin (by
adjusting pH). At pH < pi of the protein the microwave effect is diminished, while at pH
> pi the nonthermal effect is amplified. This may indicate that salt bridges or water-ion
interaction may also play a pivotal role in compensating for the nonthermal effects o f
microwave irradiation.
Heating P-lactoglobulin solutions at 75 °C by microwave irradiation and conventional
heating revealed substantial changes in the secondary structure o f P-lactoglobulin;
however, nonthermal effect o f microwave irradiation resulted in more extensive
aggregation of the protein in comparison to conventional heating effects.
The results also showed that sequence o f the unfolding pathway up on heating Plg thermally before H-D exchanges takes place started by loosening the P-sheet that are
inaccessible to solvent first, followed by the unfolding o f the solvent exposed P-sheet.
The unfolding pathways o f both microwave irradiated and conventionally heated samples
of fully H-D exchanged P-lg temperature (35°C) were the same. This illustrated that the
nonthermal effects o f microwave irradiation do not alter the unfolding pathway o f the
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protein but may in effect lower the activation energy o f the unfolding process by subtle
modifications o f the protein structure.
The unfolding pathway o f (3-lg started with an increase in the random coil,
followed by decease in the intramolecular P-sheet content culminating in the formation of
intermolecular p-sheet. The extent o f p-lg intermolecular p-sheet formation was
significantly higher as a result of the nonthermal effect o f microwave irradiation on p-lg
in solution.
The nonthermal effect of microwave irradiation on human hemoglobin (Hb) was
minimal at temperatures between 25 to 48°C. At 54°C a significant nonthermal effect on
the tertiary and secondary structure was observed. The nonthermal effect increases the
solvent exposure to the interior o f the Hb.
The amount o f protein unfolding also
increased as evidenced by the formation o f a higher population o f intermolecular P-sheet.
As the number o f cycle increased, the microwave effect was progressively amplified. This
indicates that the nonthermal effect is cumulative and that the protein retains a “memory”
of the nonthermal effect with repeated cycles.
A possible mechanism of the nonthermal microwave effect on may involve the
direct interaction o f microwave energy with the protein backbone leading to loosening of
the tertiary structure. At higher irradiation temperatures this may enhance the
intermolecular entanglement between the proteins yielding extensive aggregation.
110
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