close

Вход

Забыли?

вход по аккаунту

?

206

код для вставкиСкачать
Journal of the Science of Food and Agriculture
J Sci Food Agric 80:447±452 (2000)
Protein stability function relations: native
b-lactoglobulin sulphhydryl–disulphide
exchange with PDS
Richard K Owusu Apenten* and Despina Galani†
Laboratory of Food Biochemistry and Nutrition, Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK
Abstract: Intermolecular sulphhydryl±disulphide exchange with b-lactoglobulin dimer occurs when
this dissociates to form monomers exposing two SH groups. This notion is re-evaluated in the light of
recent structural data suggesting that the degree of SH group exposure in b-lactoglobulin is unaffected
by dissociation. b-Lactoglobulin was treated with 2,2'-dipyridyl disulphide (PDS). The rate of
sulphhydryl±disulphide exchange was measured at sub-denaturation temperatures of 25±60 ° C.
Parallel studies were conducted by reacting PDS with reduced glutathione (GSH). The SH group of
GSH was up to 31 000 times more reactive than b-lactoglobulin. At pH 7 the reaction activation
enthalpy (DH#) and entropy (DS#) was 26 kJ molÿ1 and ÿ100 J molÿ1 Kÿ1 respectively for GSH. For blactoglobulin, DH# was 157.2 kJ molÿ1 and DS# was 254 J molÿ1 Kÿ1. At pH 2.6, DH# was 14.4 kJ molÿ1 and
DS# was ÿ213 J molÿ1 Kÿ1 for GSH. The corresponding results for b-lactoglobulin were 20.3 kJ molÿ1 and
ÿ147 J molÿ1 Kÿ1. These and other thermodynamic results are discussed in terms of the effects of blactoglobulin conformational structure and stability on SH group reactivity. For native b-lactoglobulin
at neutral pH, intermolecular sulphhydryl±disulphide exchange appears to involve the dissociated
monomer. SH group activation probably arises from the lower structural stability of the monomer
relative to the dimer. At pH 2.6 the mechanism of SH±disulphide exchange does not require protein
dissociation and probably involves breathing motions or localised changes in protein structure.
# 2000 Society of Chemical Industry
Keywords: b-lactoglobulin; stability; dissociation; sulphhydryl group; disulphide exchange
INTRODUCTION
Intermolecular sulphhydryl-disulphide exchange with
b-lactoglobulin dimer occurs when this dissociates to
form monomers exposing two SH groups. This notion
needs re-examining owing to recent structural data
which suggest that the degree of SH group exposure in
b-lactoglobulin is unaffected by dissociation (see
below). This is an important issue, since the sulphhydryl (SH) groups of b-lactoglobulin dimer affect many
functional properties of this protein.
Studies employing sulphhydryl-blocking agents led
to the realisation that SH±disulphide exchange is
implicated in heat gelation and texturisation of blactoglobulin and other milk proteins.1±7 SH±disulphide exchange is engaged in the formation of a blactoglobulin±k casein complex.8±10 The SH group
concentration in milk decreases upon heating owing to
SH±disulphide interchange.11,12 Oxidation of SH
groups by iodate reduces deposit formation during
ultrahigh-temperature (UHT) treatment of milk.13
Mottram et al 14 demonstrated that changes in the
¯avour threshold of disulphide compounds such as
bis-2-furanylmethyl disulphide may be due to SH±
disulphide interchange with proteins.
SH±disulphide exchange stabilises adsorbed blactoglobulin ®lms, and this process is important for
surface functional properties such as emulsi®cation,15±17 foaming18 and fouling of process equipment.19 Adsorption at an interface provides the
surface energy for b-lactoglobulin denaturation, SH
group exposure and SH±disulphide exchange. Highpressure treatment induces a b-lactoglobulin conformational change and aggregation partly by SH±
disulphide exchange.20,21 SH group exposure increases the antioxidant effect of b-lactoglobulin
against the oxidation of linoleic acid.22,23 Ureainduced gelation of b-lactoglobulin also occurs via
SH±disulphide exchange.24
The connection between protein structure and SH
group reactivity has been emphasised in the past. SH
group activation can be traced to changes in protein
conformation brought about by surface, temperature,
pressure or urea denaturation. The conformational
control of SH group reactivity can be understood if
* Correspondence to: RK Owusu Apenten, Laboratory of Food Biochemistry and Nutrition, Procter Department of Food Science, University of
Leeds, Leeds LS2 9JT, UK
E-mail: r.k.owusu@leeds.ac.uk
†
Current address: Cambridge University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 1EW, UK
Contract/grant sponsor: Hellenic State Scholarship Foundation
(Received 12 April 1999; revised version received 24 August 1999; accepted 29 October 1999)
# 2000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2000/$17.50
447
RKO Apenten, D Galani
such groups are masked within native proteins. Hence
8 M urea is recommended as solvent during the
quantitative determination of protein SH groups.25±27
SH group reactivity has not been fully investigated for
non-enzymatic proteins in their native state. Functions
such as metal ion binding, responses to oxidative stress
and protein±protein interactions may involve SH
groups.28±30
Kinetic measurements can provide insights concerning the environment of protein SH groups.31
Native b-lactoglobulin was reacted with Ellman's
reagent. The kinetics of the reaction led to suggestions
that SH group reactivity was moderated by protein
dissociation. The SH groups of b-lactoglobulin dimer
became exposed to solvent after dissociation leading to
their activation.32 However, recent X-ray crystallographic data33 con®rm the results of earlier chemical
studies.34 In b-lactoglobulin monomer the SH group is
not solvent-accessible. In the light of such results,
factors that affect SH group reactivity for native blactoglobulin dimer should be re-examined.
The results of our investigations in this area are
presented in this paper. b-Lactoglobulin was treated
with 2,2'-dithiodipyridine (PDS), a low-molecularweight disulphide compound. Unlike Ellman's reagent, PDS can be used at low pH.35 The rate of SH±
disulphide exchange was determined from spectrophotometric measurements at pH 7 and 2.6 and at
temperatures (25±60 °C) below those that denature blactoglobulin. Using PDS, SH reactivity was measured
under conditions where b-lactoglobulin exists as the
monomer (see below). Parallel studies were performed
with glutathione (GSH), a simple tripeptide having a
solvent-accessible SH group. Activation free energy
(DG#), enthalpy (DH#) and entropy (DS#) values were
determined from the temperature dependence of the
rates of reaction for both b-lactoglobulin and GSH.
The results are discussed in terms of the possible
effect of protein stability on their functional properties.36,37 SH group activation may arise from a
reduction in the stability of the b-lactoglobulin
monomer relative to the dimer.
(PDS) and 2-thiopyridine (2-TP) were from Sigma
Aldrich Co Ltd (Poole, UK).
Measurement of rate of sulphhydryl–disulphide
exchange
The rate of SH±disulphide exchange was measured in
0.1 M potassium phosphate buffer pH 7 or 0.1 M
glycine±glycine HCl buffer pH 2.6. PDS (2.2 mg)
was dissolved in 5 ml of buffer by stirring for several
hours at room temperature. Insoluble material was
removed by ®ltration. The PDS stock solution (1.0±
1.5 mM) was diluted and the exact concentration was
determined from absorbance readings at 281 nm
(ε281 = 9730 M-1 cm-1).39 The instrument used was a
Pye Unicam (model SP-800) twin-beam UV-vis
spectrophotometer ®tted with a thermostated cuvette
holder. All buffers contained 0.02% sodium azide as
preservative.
SH±disulphide exchange was initiated by adding blactoglobulin or GSH (0.3 ml; 2.2 10ÿ4 M ®nal
concentration) to PDS (2.7 ml; 2.5 10ÿ4 M ®nal
concentration). Absorption readings were taken at
343 nm (A343) with time. The reference cuvette
contained 2.7 ml of PDS solution plus 0.3 ml of buffer.
The reaction temperature was controlled using a
circulating water bath. If C0 is the initial concentration
of b-lactoglobulin or GSH (MWt = 36 800 for blactoglobulin and 307.3 for GSH) and Ct is the
remaining free SH group concentration at various
timed intervals, then Ct = C0 ÿCx, where Cx = A343/
ε343. Kinetic data were analysed according to secondorder kinetics.35,39 All rate measurements were performed in triplicate.
RESULTS AND DISCUSSION
SH±disulphide exchange between GSH and PDS is
described by the following equation, where I refers to
PDS, II is GSH, III is a mixed disulphide product and
IV is 2-thiopyridine (2-TP):
Ar-SS-Ar ‡ XSH ! Ar-SS-X ‡ Ar-SH
‰IŠ
EXPERIMENTAL
Preparation of b-lactoglobulin
b-Lactoglobulin was prepared by ammonium sulphate
fractionation38 and puri®ed by preparative-scale gel
®ltration chromatography on a Superdex-75 matrix
(2.6 cm 100 cm, XK 26/100, Pharmacia Biotech)
using a Gradi Frac system (Pharmacia). The purity of
the b-lactoglobulin was con®rmed by FPLC±gel
®ltration (Superdex 75), FPLC±anion exchange
(Mono-Q HR 5/5) chromatography and sodium
dodecyl sulphate polyacrylamide gel (SDS-PAGE)
chromatography. Freeze-dried b-lactoglobulin samples contained 85% w/w protein (remainder being
salts), of which 99% was b-lactoglobulin as determined by SDS-PAGE. Glutathione (GSH; reduced
form), dipyridyl disulphide or 2,2'-dithiodipyridine
448
‰IIŠ
‰IIIŠ
…1†
‰IVŠ
At ®rst, 2-mercaptopyridine is formed which tautomerises to give 2-TP. Consequently, SH±disulphide
exchange with PDS is irreversible. The concentration
of 2-TP was obtained from A343 readings as described
above.27,35,39 The molar absorption coef®cient for 2TP was determined, by taking A343 readings from
standard solutions of 2-TP, as 7076 Mÿ1 cmÿ1 at pH
2.6±8.4 and at temperatures of 25±60 °C.
For native globular proteins the reaction in eqn (1)
occurs after a conformational change to expose any
buried SH group.36 In this study, protein±protein SH±
disulphide exchange did not occur. b-Lactoglobulin
polymers were not found when samples were analysed
by FPLC±gel permeation chromatography. From the
FPLC elution volume, chemically modi®ed b-lactoglobulin had a molecular weight of 18.4 kDa. UnJ Sci Food Agric 80:447±452 (2000)
b-Lactoglobulin stability and SH group reactivity
The quantity [IV] is obtained from A343 measurements at timed intervals, whence
d‰IVŠ=dt ˆ k…‰IŠ0 ÿ ‰IVŠ†…‰IIŠ0 ÿ ‰IVŠ†
…3†
After integration, eqn (3) gives
1=…‰IŠ0 ÿ ‰IIŠ0 † lnf…‰IŠ0 ÿ ‰IVŠ†‰IIŠ0 =
…‰IIŠ0 ÿ ‰IVŠ†‰IŠ0 g ˆ kt
…4†
A plot of the left-hand side of eqn (4) versus time t gave
a straight-line graph (not shown), the gradient being
the rate constant k (M-1 s-1).
Activation parameters for SH–disulphide exchange
The activation free energy change DG# for SH±
disulphide exchange is expressed as
DG# ˆ ÿRT ln…kh=KT †
…5†
where k is the rate constant, h is the Planck constant
and K is the Boltzmann constant. Combining this with
DG# = DH# ÿTDS# leads to
k
DH #
h
DS #
‡ ln
…6†
ln
ˆÿ
‡
RT
R
T
K
Figure 1. Arrhenius plots of the SH–disulphide exchange reaction for blactoglobulin‡ PDS and glutathione‡ PDS. (a) pH 7; (b) pH 2.6. All points
are the mean of triplicate measurements.
modi®ed b-lactoglobulin had a molecular weight of
36.8 kDa. Apparently, SH group modi®cation prevents b-lactoglobulin dimer formation.40,41
From eqn (1) the rate of reaction is d[IV]/dt = k [I]
[II]. For initial concentrations [I]0 and [II]0,
‰IŠ ˆ ‰IŠ0 ÿ ‰IVŠ and
‰IIŠ ˆ ‰IIŠ0 ÿ ‰IVŠ
J Sci Food Agric 80:447±452 (2000)
…2†
We then plotted ln(k/T) vs 1/T. The slope of this graph
is DH#/R and the intercept is ln(h/K) ‡ DS#/R (Figs
1(a) and 1(b)). Values for activation parameters
determined using eqn (6) are reported in Table 1.
The denaturation temperature (Td) for b-lactoglobulin is 70.5 0.14 °C under the conditions used in
this study.42 The exact value of Td depends on protein
concentration, pH and solvent ionic strength.43 At
sub-denaturation temperatures, DH# and DS# values
for SH±disulphide exchange with b-lactoglobulin can
be ascribed to a rate-limiting reaction involving a
protein conformational change. In contrast, DH# and
DS# for the GSH reaction are typical of values
associated with simple chemical reactions.37,44,45
Beyond such generalisations, absolute values for DH#
and DS# are not easy to interpret owing to the entropy±
enthalpy compensation phenomenon.46 The magnitudes of DH# and DS# are not considered further.
The b-lactoglobulin reaction with PDS is a secondorder, unimolecular process at pH 7. The order of a
reaction (the x-term in the rate equation: rate = k[C]x)
is not related to the number of molecules involved in
formation of TS*, ie the `molecularity'.47 The PDS
reaction with GSH proceeds via a second-order,
bimolecular, nucleophilic substitution (SN2). The
rate-liming step involves a collision of GSH and PDS
molecules. As evidence for an SN2 process, values for
DS# were negative. The SN2 reaction is summarised
as48
GS
#
GSÿ ‡ Ar.S-S.Ar ! ‰Ar-S-S-ArŠ
…7†
! Ar-S-S-G ‡ Ar.Sÿ
449
RKO Apenten, D Galani
System
Table 1. Activation parameters for
sulphhydryl–disulphide exchange
between PDS and b-lactoglobulin or
glutathione
b-Lactoglobulin pH 7 (25±60 °C)
Glutathione pH 7 (10±25 °C)
b-Lactoglobulin pH 2.6 (25±60 °C)
Glutathione pH 2.6 (25±50 °C)
a
DS
(J molÿ1 Kÿ1)
157.3 (0.1)
26.0 (0.06)
14.4 (0.08)
20.3 (0.05)
254 (1.6)
ÿ100 (0.43)
ÿ213 (2.1)
ÿ147 (2.4)
DG#
(kJ molÿ1) at 25 °C a
81.5
55.8
78.8
64.6
Coef®cient of variation for rate measurements from which DG# values are calculated is 3.7% (n = 3 replicates).
where GSÿ is deprotonated GSH and the TS* is in
square brackets. Formation of a TS* from two
reactants accounts for the negative activation entropy.47
On the mechanism of SH group activation for native
b-lactoglobulin
DG# provides a clear index of the energy cost for
producing TS* (Table 1). Values for DG# were greater
for b-lactoglobulin compared with GSH by 25.7 and
14.2 kJ molÿ1 at pH 7 and 2.6 respectively.
Differences in DG# for b-lactoglobulin compared
with GSH (DDG# values) depend on the respective
environments of the SH groups. Glutathione is a
tripeptide (Gly.Cys.Glu) which functions in maintaining the intracellular redox potential.49 Owing to its
small size, the structure of GSH will not shield its SH
group from the solvent. By comparison, b-lactoglobulin is a globular protein which, at physiological pH,
exists as a dimer.40,50,51 The SH groups of blactoglobulin are not accessible to the external solvent.
Below pH 3.5 the dimer dissociates to form monomers
owing to electrostatic repulsion between the two
subunits.51
X-ray crystallography results show that b-lactoglobulin has nine b-sheets (strands A, B, C, D, E, F, G, H
and I) and a short a-helix. The 3 ° structure is a shallow
cone. Two disulphide links occur at Cys66±Cys160
and Cys106±Cys19. One SH group per monomer
occurs at Cys121 far from the subunit±subunit
interacting interface.33 Dissociation of b-lactoglobulin
dimer is therefore unlikely to expose the SH group to
the external solvent.33,34
SH±disulphide exchange with PDS at pH 2.6
required some sort of conformational change in the
b-lactoglobulin (monomer). Whatever the nature of
this structural change, it occurs under non-denaturing
conditions. The high DG# values for b-lactoglobulin
(compared with GSH; Table 1) support this notion.
This behaviour should be distinguished from that seen
under denaturing conditions. Then b-lactoglobulin
unfolds, thereby increasing its SH group reactivity.2,52
Under denaturing conditions, SH group activation
coincides with unfolding of the backbone structure for
b-lactoglobulin in 4±8 M urea.37
Despite current structural data, SH group activation
is somehow linked to the dissociation of b-lactoglobulin dimer.32 Hence DDG# was 25.7 kJ molÿ1 at pH 7
(Table 1) and very nearly identical to the free energy
change (26 kJ molÿ1) for b-lactoglobulin dissocia450
DH#
(kJ molÿ1)
tion.40,53±55 Also, modifying the SH group of blactoglobulin prevents re-dimerisation of the subunits.40,41 Finally, SH group activation and dissociation
occur at the same concentrations (0±3 M) of urea.32
Here then is the paradox. From its location, Cys 121
is not likely to be exposed by the dissociation of blactoglobulin.33,34 However, there is apparently good
evidence linking dissociation and SH group activation.32
A model for SH group activation
The above consideration leads us to suggest that
perhaps the link between b-lactoglobulin dissociation
and SH group activation is not one of geometry. It may
be supposed that SH group activation involves `closed'
and `open' forms of b-lactoglobulin in `rapid' equilibrium, the open form (not necessarily a monomer)
having an exposed/reactive SH group:
closed „ open ! SH--disulphide exchange
…8†
Furthermore, it may be supposed that DDG# is the
energy change associated with a closed „ open
transition. The equilibrium constant from eqn (8)
(Keq = [open]/[closed]) can be estimated from
DDG# = ÿRT ln Keq. Therefore, with DDG# =
25.7 kJ molÿ1 (Table 1; pH 7), Keq = 3.21 10ÿ7.
Under the non-denaturing conditions the concentration of the `open' form is 3.12 10ÿ5% at neutral
pH.37 This concentration of `open' molecules is
suf®cient to account for the SH group reactivity
observed with the native protein. However, the low
concentration of the open form would make it dif®cult
to characterise this state in the presence of a large
excess of the closed form. The rate of SH±disulphide
exchange also decreases for `dry' b-lactoglobulin
crystals. In conclusion, the degree of solvent exposure
needed to account for SH group activation in native blactoglobulin may be too `small' for direct analysis
using current physical methods.
SH group activation is also possible owing to
changes in protein stability.36,37 In brief, the SH group
of b-lactoglobulin will be activated by dissociation if
this event produces a reduction of the stability of
native b-lactoglobulin. Native proteins are in constant
motion about an average set of co-ordinates.56 The
frequency of such transitions is related to the
conformational stability.37 If dissociation of b-lactoglobulin leads to a 26 kJ molÿ1 reduction in its stability,
then, as compared with the dimer form, the monomer
J Sci Food Agric 80:447±452 (2000)
b-Lactoglobulin stability and SH group reactivity
will be 32 000 times more ¯exible and thus more likely
to expose Cys121 to solvent. The effect of the 4 °
structure on SH group activity is not solely determined
by the location of Cys121. It is proposed that subunit
dissociation leads to enhanced reactivity of the SH
group of b-lactoglobulin owing to the effect on protein
global ¯exibility. The unfolding stability of b-lactoglobulin is being studied.57
There are several indications that, at low pH, blactoglobulin SH group reactivity is not dependent on
the dissociation of protein dimer. The transition from
a `closed' to an `open' form (eqn (8)) is not a
dissociation process. First, the dissociation free energy
has been reported as 22.6 kJ molÿ1 for b-lactoglobulin
at pH 2.6.51,53 On the other hand, DDG# from the
present study was 14.2 kJ molÿ1 at pH 2.6. The free
energy for exposing an SH group is apparently lower
than the free energy change for dissociation. Second,
applying the former principles to results at pH 2.6,
with DDG# equal to 14.2 kJ molÿ1, then Keq =
3.24 10ÿ3 and the concentration of the `open' form
of b-lactoglobulin is 3 10ÿ3%. This is 90 times
greater than the concentration found at pH 7. On
the other hand, the extent of protein dissociation at pH
2.6 is only twofold greater than the degree of
dissociation at pH 7. For a protein concentration of
8±10 mg mlÿ1, b-lactoglobulin was 20% and 40%
dissociated at pH 7 and 2.6 respectively.57 The
simplest hypothesis that accounts for the results at
low pH is that SH±disulphide exchange with PDS
takes place from b-lactoglobulin dimer. A nondissociation mechanism could involve breathing-type
motions or localised conformational transitions in the
intact dimer.
ACKNOWLEDGEMENTS
We thank the Hellenic State Scholarship Foundation
for a scholarship to DG, and Dr C Holt (Hannah
Research Institute, Ayr, UK) for advice on the
preparation of b-lactoglobulin.
REFERENCES
1 Sawyer WH, Heat denaturation of bovine beta-lactoglobulin and
relevance of disul®de aggregation. J Dairy Sci 51:323±329
(1967).
2 Franklin JO and Leslie J, The kinetics of the reaction of Nethylmaleimide with denatured b-lactoglobulin and ovalbumin. Biochim Biophys Acta 160:333±339 (1968).
3 Watanabe K and Klostermeyer H, Heat induced changes in
sulfhydryl and disul®de levels of b-lactoglobulin-A and the
formation of polymers. J Dairy Res 43:411±418 (1976).
4 Mangino ME, Kim JH, Dunkerley JA and Zadow JO, Factors
important to the gelation of whey protein concentrates. Food
Hydrocolloids 1:277±282 (1987).
5 Hashizume K and Sato T, Gel-forming characteristics of milk
proteins. II. Roles of sulfhydryl groups and disul®de bonds. J
Dairy Sci 71:1447±1454 (1988).
6 Shimada K and Cheftel JC, Texture characteristics, protein
solubility, and sulfhydryl group/disul®de bond contents of
heat-induced gels of whey protein isolate. J Agric Food Chem
36:1018±1025 (1988).
J Sci Food Agric 80:447±452 (2000)
7 Hoffman MAM and Van Mil PJJM, Heat-induced aggregation
of beta-lactoglobulin. Role of the free thiol group and disul®de
bonds. J Agric Food Chem 45:2942±2948 (1997).
8 Kinsella JE, The chemistry of dairy powders with reference to
baking. Adv Food Res 19:147±213 (1971).
9 Haque Z, Kristjiansson MM and Kinsella JE, Interaction
between kappa-casein and b-lactoglobulin: a possible mechanism. J Agric Food Chem 35:644±649 (1987).
10 Hill AR, The b-lactoglobulin±kappa-casein complex. Can Inst
Food Sci J 22:120±123 (1989).
11 Patrick PS and Swaisgood HE, Sulfhydryl and disul®de groups in
skim milk as affected by direct ultra-high-temperature heating
and subsequent storage. J Dairy Sci 59:594±600 (1976).
12 Parnell-Clunies E, Kakuda Y and Irvine D, Heat-induced
protein changes in milk processed by vat and continuous
heating systems. J Dairy Sci 71:1472±1483 (1988).
13 Skudder PJ, Thomas EL, Pavey JA and Perkin AJ, Effects of
adding potassium iodate to milk before UHT treatment. I.
Reduction in the amount of deposit on the heated surfaces. J
Dairy Res 48:99±113 (1988).
14 Mottram DS, Szauman-Szumski C and Dodson A, Interactions
of thiol and disul®de ¯avour compounds with food components. J Agric Food Chem 44:2349±2351 (1996).
15 Courthaudon JL, Dickinson E, Matsumura Y and Williams A,
In¯uence of emulsi®er on the competitive adsorption of whey
proteins in emulsions. Food Struct 10:109±115 (1991).
16 McClements DJ, Monahan FJ and Kinsella JE, Disul®de bond
formation affects stability of whey protein isolate emulsions. J
Food Sci 58:1036±1039 (1993).
17 Monahan FJ, McClements DJ and German JB, Disul®demediated polymerisation reactions and physical properties of
heated WPI stabilised emulsions. J Food Sci 61:504±509
(1996).
18 Liao SY and Mangino ME, Characterisation of the composition,
physicochemical and functional properties of acid whey
protein concentrates. J Food Sci 52:1033±1037 (1987).
19 Roscoe SO, Fuller KL and Robitaille O, An electrochemical
study of the effect of temperature on the adsorption behaviour
of beta-lactoglobulin. J Colloid Interface Sci 160:243±251
(1993).
20 Galazka VB, Sumner IO and Ledward DA, Changes in protein±
protein and protein±polysaccharide interactions induced by
high pressure. Food Chem 57:393±398 (1996).
21 Funtenberger S, Dumay E and Che®el JC, High pressure
promotes b-lactoglobulin aggregation through SH/S-S interchange reactions. J Agric Food Chem 45:912±921 (1997).
22 Taylor MJ and Richardson T, Antioxidant activity of skim milk:
effect of heat and resultant sulfhydryl groups. J Dairy Sci
63:1783±1795 (1980).
23 Moller RE, Stapelfeld H and Skibsted LH, Thiol reactivity in
pressure-unfolded beta-lactoglobulin. Antioxidative properties
and thermal refolding. J Agric Food Chem 46:425±430 (1998).
24 Xiong YL and Kinsella JE, Mechanism of urea-induced whey
protein gelation. J Agric Food Chem 38:1887±1891 (1990).
25 Manning PB, Heinselman AL, Jenness R and Coulter ST,
Method for determining sulfhydryl and disul®de groups in
milk proteins. J Dairy Sci 52:886±887 (1969).
26 Beveridge T, Toma S and Nakai S, Determination of SH- and
SS-groups in some food proteins using Ellman's reagent. J
Food Sci 39:49±51 (1974).
27 Jocelyn PC, Spectrophotometric assay of thiols. In Sulfur and
Sulfur Amino Acids, Vol 143 of Methods in Enzymology, Ed by
Jakoby WB and Grif®th W, Academic Press, London, pp 45±
67 (1987).
28 Jocelyn PC, Biochemistry of the SH-group. The Occurrence,
Chemical Properties, Metabolism and Biological Function of Thiols
and Disul®des, Academic Press, London (1972).
29 Friedman M, Improvement in the safety of foods by SHcontaining amino acids and peptides. A review. J Agric Food
Chem 42:2±20 (1994).
30 Packer L (Ed), Biothiols. Part A. Monothiols and Dithiols, Protein
451
RKO Apenten, D Galani
31
32
33
34
35
36
37
38
39
40
41
42
43
Thiols and Thiyl Radicals, Vol 251 of Methods in Enzymology,
Academic Press, New York (1995).
Halasz P and Polgar L, Effect of the immediate environment on
the reactivity of the essential SH group of papain. Eur J
Biochem 71:571±575 (1976).
Kella NKD and Kinsella JE, Structural stability of b-lactoglobulin in the presence of kosmotropic salts. A kinetic and
thermodynamic study. Int J Peptide Protein Res 32:396±405
(1988).
Brownlow S, Morais Cabral JH, Cooper R, Flower DR, Yerdall
SJ, Polikarpov I, North ACT and Sawyer L, Bovine bÊ resolutionÐstill an enigmatic lipocalin.
lactoglobulin at 1.8 A
Structure 5:481±495 (1997).
Townend R, Herskovits TT and Timasheff SS, The state of
amino acid residues in b-lactoglobulin. Arch Biochem Biophys
129:567±580 (1969).
Grasseti DR and Murray JF, Determination of sulfhydryl groups
with 2,2'- or 4,4'-dithiodipyridine. Arch Biochem Biophys
119:41±49 (1967).
Apenten RKO, Protein stability function relations: b-lactoglobulin-A sulfhydryl group reactivity and its relationship to
protein unfolding stability. Int J Biol Macromol 23:19±25
(1998).
Apenten RKO, The effect of protein unfolding stability on their
rates of irreversible denaturation. Food Hydrocolloids 12:1±8
(1998).
Armstrong JMCD, Hooper KE, Mckenzie HA and Murphy
WH, On the column chromatography of bovine whey proteins.
Biochim Biophys Acta 214:419±426 (1970).
Pedersen AO and Jacobsen J, Reactivity of the thiol group in
human and bovine albumin at pH 3±9, as measured by
exchange with 2,2'-dithiodipyridine. Eur J Biochem 106:291±
295 (1980).
Zimmerman JK, Barlow OH and Klotz IM, Dissociation of blactoglobulin near neutral pH. Arch Biochem Biophys 138:101±
109 (1970).
Iametti I, de Gregori B, Vecchio G and Benomi F, Modi®cations
occur at different structural levels during heat denaturation of
b-lactoglobulin. Eur J Biochem 237:106±112 (1996).
Galani D, A study on the thermal denaturation of bovine betalactoglobulin. PhD Thesis, University of Leeds (1999).
Qi XL, Brownlow S, Holt C and Sellers P, Thermal denaturation
452
44
45
46
47
48
49
50
51
52
53
54
55
56
57
of b-lactoglobulin: effect of protein concentration at pH 6.75
and 8.05. Biochim Biophys Acta 1248:43±49 (1995).
Owusu RK and Bethalon N, A test for the two-stage thermoinactivation model for chymotrypsin. Food Chem 48:231±235
(1993).
Apenten RKO and Berthalon N, Determination of enzyme
global thermostability from equilibrium and kinetic analysis of
heat inactivation. Food Chem 51:15±20 (1994).
Forsyth JL, Apenten RKO and Robinson DS, The thermostability of puri®ed isoperoxidases from Brassica oleracea Var.
gemmifera. Food Chem 65:99±109 (1999).
Pilling MJ and Seakins PW, Reaction Kinetics, Oxford University
Press, Oxford (1995).
Malthouse JPG and Brocklehurst K, A kinetic method for the
study of solvent environments of thiol groups in proteins
involving the use of a pair of isomeric reactivity probes and a
differential solvent effect. Biochem J 185:217±222 (1980).
Stryer L, Biochemistry, 4th edn, WH Freeman, New York, pp
568, 731 (1996).
Mckenzie HA and Sawyer WH, Effect of pH on b-lactoglobulin.
Nature 214:1101±1104 (1967).
Timasheff SN and Townend R, Structural and genetic implications of the physical and chemical differences between blactoglobulin A and B. J Dairy Sci 45:259±266 (1962).
Dunnill P and Green DW, Sulfhydryl groups and NIR
conformational change in b-lactoglobulin. J Mol Biol 15:147±
151 (1965).
Kelly MJ and Reithel FJ, A thermodynamic analysis of the
monomer±dimer association of b-lactoglobulin A at the
isoelectric point. Biochemistry 10:2639±2644 (1971).
Aymard P, Durand D and Nicolai T, The effect of temperature
and ionic strength on the dimerisation of b-lactoglobulin. Int J
Biol Macromol 19:213±221 (1996).
Joss LA and Ralston GB, b-Lactoglobulin B: a proposed
standard for the study of reversible self-association reaction
in an analytical ultracentrifuge? Anal Biochem 236:20±26
(1996).
Creighton TE, Proteins: Structure and Molecular Properties, WH
Freeman, New York (1984).
Galani D and Apenten RKO, b-lactoglobulin denaturation by
dissociation-coupled unfolding. Food Res Int 32:93±100
(1999).
J Sci Food Agric 80:447±452 (2000)
Документ
Категория
Без категории
Просмотров
5
Размер файла
107 Кб
Теги
206
1/--страниц
Пожаловаться на содержимое документа