Food Hydrocolloids Vol. 11 no . 2 pp.

171-180, 1997
-
Thermodynamic incompatibility of proteins

V.1.Polyakov l ,4, V.Ya.Grmberg! and V.B.Tolsteguzov-

llnstitute of Biochemical Physics, Russian Academy of Sciences, Vavilov Str. 28, 117813 Moscow aSP-I , Russia and 2Nestec
Research Centre, Verz-chez-les-Blanc, PO Box 44, CH 1000, Lausanne 26, Switzerland
4To whom correspondence should be addressed

Abstract
Unlike mixtures of synthetic polymers, mixtures of polysaccharides and protein-polysaccharide
mixtures, the thermodynamic incompatibility of proteins in solution is notable for the following general
features. This phenomenon is typical of mixtures of proteins belonging to different classes according to
the Osborne classification. A relatively high phase separation threshold is the other characteristic feature
of mixed protein solutions. Factors such as p H, ionic strength and temperature only slightly affect
protein incompatibility. This is probably due to the compactness and rigidity of protein molecules.
Accordingly, incompatibility is strongly dependent upon the conformational state of the proteins and is
enhanced by protein denaturation.

Introduction
Most foods contain mixtures of proteins. Food proteins
perform two main functions, nutritional and structural, that
are of great importance for food quality. Relative to these
two functions, a mixture of food proteins can display both
synergistic and antagonistic effects. Since protein mixtures
often have a higher nutritional value and better functional
properties compared to individua l protein components,
protein additives are widely used for controlling the
nutritional value and functional properties of food systems.
The contribution of a protein additive to nutritional quality
can be calculated from its amino acid composition and that
of the food system. However, this is not the case for
structural functions of a food protein, where an empirical
approach is usually used for controlling functional properties
of food systems with added proteins. Until recently, many
fundamental aspects of protein behaviour as they concern
structural functions of a food protein in a given food system
were completely unknown. For instance, it was not clear
Whether proteins could be mixed on a molecular level and the
thermodynamic properties of proteins in their mixtures were
unknown . To understand and develop novel food processes
and products, it is necessary to study phase behaviour and
the structure-property relationship of mixed protein
Solutions. This is also of importance for the stability of
dispersed food systems stab ilized by proteins, in particular
against depletion flocculation. In spite of the generalized
practical use of protein mixtures, the thermodynamic
behaviour of proteins remained unstudied before 1979 (1-6).
Only some data on the osmotic pressure of dilute mixed
protein solutions (7-11) as well as data concerning the
swelling behaviour of gelatin gels filled by globular proteins
(12) were available. It could be assumed, based on these data,
that proteins might be thermodynamically incompatible in
solution. The osmotic pressure of a mixed protein solution
was found to be higher than the sum of osmotic pressures of
the individual proteins under comparable conditions (7-11).
This means that the cross second virial coefficient that
characterizes intermolecular interactions between different
proteins may exceed the second virial coefficients reflecting
interactions between molecules of each individual protein.
Th is relationship between the virial coefficients is typical of
thermodynamic incompatibility of solutes of ternary
systems (13). The next indirect evidence for thermodynamic
incompatibility of proteins was the fact that gelatin gels filled
with globular proteins swell more strongly than gels of pure
gelatin (12). Nevertheless, the problem of thermodynamic
incompatibility of proteins was not considered (7-12).
It should be noted that thermodynamic incompatibility of
proteins is rather closely related to the problem of
fractionation of proteins according to their solubility, a
subject that has been extensively discussed (14-17). However,
in the case of protein incompatibility, phase separation
usually leads to two liquid phases. By contrast, precipitation
of a protein fraction usually results in the formation of

ClOXford University Press

 172 V I. Polyakov, V Ya. Grinberg and VB. Tolstoguzov

liquid and solid (crystalline or amorphous) phases. The main Table J Incompatibility conditions for some protein I-protein
features of liquid-liquid and liquid-solid phase equilibria 2-water systems
are different (18).
No. Systems Incompatibility conditions Refs
Systematic study of the thermodynamic incompatibility of
proteins has been carried out at the Institute of I Albumin-albumin:
Organo-Element Compounds in Moscow. It was found that 1.1. Ovalbumin (OA)-BSA not found at pH 2-10 and la 23
many proteins are thermodynamically incompatible in s 1 (NaCI); 25°C
aqueous solutions. The phase behaviour of mixed protein 1.2. OA-oATC*·b pH 6.7; water; 20°C 24
solutions has also been studied (19-25). The main goal of 1.3. BSA-oATC* pH 6.7; water; 20°C 24
this paper is to consider the specific features of thermo- 104. BSA-gelatin pH 7.8; I s 1 (NaCI); 40°C 18
dynamic incompatibility of proteins.
II. Globulin-globulin:
ILL Fibrinogen-SB globulin pH 7.9; I = 004 (NaCl); 25°C 18
fraction''
Generality of the phenomenon of thermodynamic
incompatibility of proteins III Albumin-globulin:
The proteins studied are given in Table 1 according to the III. 1. OA-glycinind pH 6.8; water; 20°C 44
Osborne classification (26) based on protein solubility, i.e. 111.2. OA-SB globulin fraction* pH 6.6; water; 20°C 20
albumins, globulins, glutelins and prolamins. This I1I.3. BSA-fibrinogen pH 6.6; water; 20°C 18
classification reflecting protein-solvent interactions is IlIA. BSA-glycinin pH 6.7; water; 20°C 44
thermodynamic by nature and probably the most relevant for III.5. BSA-SB globulin fraction pH 4.9; I = 0.39 (NaCl); 18
discussion of protein incompatibility. Table 1 shows that only 25°C
one exception was found among the 19 protein I-protein 111.6. Gelatin-legumin*,e pH 7.0; water; 40°C 22
2-water systems studied: the ovalbumin-bovine serum III.7. Gelatin-BB globulin pH 7.0; water; 40°C 22
albumin (BSA) -water system. This system remained in fractions-"
single-phase state up to a total concentration of 50% protein
as well as during variations of pH and ionic strength (i.e. IV Albumin-prolamin:
over the whole experimentally accessible area of system IY.l. BSA-gliadin pH 11.0; water; 25°C 18
composition). This compatibility is presumably due to the
formation of weak ovalbumin-BSA complexes found by a V Albumin-other proteins
calorimetric study (24). Thus, Table 1 demonstrates the y.1. OA-easein* pH 6.6; water; 20°C 20
general nature of the incompatibility of proteins. This Y.2. BSA-easein pH 6.9; 1 = 0.5 (NaCl); 25°C 18
phenomenon is typical of proteins belonging to different
classes within the Osborne classification. Proteins of the VI Globulin-prolamin:
same class are incompatible when they differ in their VI. 1. SB globulin pH 10.5; water; 25°C 18
conformations, e.g. the native and denatured forms of the fraction-gliadin
same protein. For instance, the soluble aggregates of
thermodenatured ovalbumin are thermodynamically VII G1obulin-other proteins
incompatible with native ovalbumin (Table 1; No. 1.2.), VII. 1. Fibrinogen-easein pH 604; 1=0.41; 18
(NH4)zS04; 25°C
globular native BSA is incompatible with gelatin (which has
VII.2. G1ycinin-easein pH 6.6; water; 20°C 44
a random coil conformation) (Table 1; No. 1.4.), and the
VII.3. BB globulin pH 7.0; water; 20°C 42
globular storage proteins from soybean are incompatible
fraction-easein *
with fibrinogen (rigid rods) (Table 1; No. 11.1.).
VIlA. SB globulin pH 6.9; water; 25°C 20
fraction-easein*

Boundary conditions for thermodynamic VIII Prolamin-other proteins

incompatibility of proteins
The study of the effects of temperature, pH and ionic
strength on boundary conditions for phase separation of
mixed protein solutions does not reveal a significant
difference in phase behaviour between pairs of proteins
belonging to different classes. Protein solubility is a key
factor for incompatibility, i.e. when two proteins of different
classes are highly soluble under given conditions they should
be incompatible at sufficiently high concentrations.
The specific feature of most protein I-protein 2-water
systems is a rather high phase separation threshold, i.e. the
VIII.l. Gliadin-easein* pH 11.0; water; 25°C 20
*There is a phase diagram.
"lonic strength. bThermo-clusters of ovalbumin. dSoybean globulin
fraction. dllS soybean globulin. ellS broad bean globulin. [Broad
bean globulin fraction.
minimum total concentration of proteins at which phase
separation of their mixed solution occurs. This value is the
sum of coordinates of the separation threshold point. As a
rule, it ranges from 10 to 20%. For systems containing
gelatin, which has a random coil conformation, the phase
 Thermodynamic incompatibility ofproteins
separation threshold is slightly below 8%. The lowest known
separation threshold (-6%) is observed for the casein-
gliadin-water system at pH 11.0. It can be assumed that
under these conditions both proteins (or at least casein) have
unfolded conformations. Presumably, the higher phase
separation threshold, i.e. higher compatibility of proteins in
a solution compared with ordinary synthetic polymers, is due
to the compactness of the protein molecules.
Taking into account that phase separation results from
multiple interactions of macromolecules and takes place in
moderately concentrated polymer solutions, De Gennes (27)
has shown that the phase separation threshold of a polymer
I-polymer 2-solvent system (at least for symmetrical cases) is
approximately equal to the critical bulk concentration
corresponding to the transition from a dilute to a moderately
concentrated solution. This boundary concentration is
usually proportional to the packing density of monomer
residues in the macromolecule. Naturally, the packing
density of amino acid residues in a globular protein is higher
than that of an ordinary flexible chain polymer of random
coil conformation (28-31). Hence, it should be expected that
the boundary concentration between dilute and moderately
concentrated solutions of proteins is significantly higher
than that of ordinary synthetic polymers. It is very likely that
this is the reason why relatively high separation thresholds
are typical of most protein I-protein 2-water systems.
It should also be noted that the incompatibility is a
consequence of non-specific short-range interactions of
macromolecules. In the case of polyelectrolytes, these
interactions can be realized only if electrostatic interactions
of the macromolecules are well screened. Under the salt-free
conditions typical of the protein systems under investigation,
this can occur at a rather high total concentration of
macromolecules due to the 'self-screening' effect (32). It is
expected that this 'self-screening' wiII be observed for
compact protein molecules at a higher concentration than for
randomly coiled macromolecules. This may be an additional
factor contributing to relatively higher phase separation
thresholds of protein I-protein 2-water systems.
Factors affecting incompatibility of proteins
The following factors are mainly responsible for
incompatibility of proteins: (i) protein l-protein 2
interaction evaluated by the parameter XPRI-PR2; (ii)
Ax-effect, i.e. a difference in the hydrophilicities of proteins I
and 2 evaluated by the protein-solvent interaction
parameters XPRI -S and XPR2-S; (iii) molecular weights of the
proteins; (iv) conformation states of the proteins. In view of
the similarity in the chemical composition of most food
proteins, it can be expected that the first two factors are
correlated with one another (Fig . I). The larger the difference
in protein hydrophilicity (Ax-effect), the higher the
interaction parameter XPRI.PR2 and, accordingly, the lower
the bulk concentration of the proteins at which the phase
separation takes place. When the interaction parameters
173
N
0::
Q.
I
0::
Q.
X
Figure 1 Schematic dependence of the parameter of protein
I-protein 2 interactions (XPRI.PR2) on the difference in the
solvent-protein interaction parameters (xPRI-S, XPR2-S) for these
proteins.
XPRI -S and XPR2-S approach each other, i.e. the Ax-effect
approaches zero, proteins I and 2 lose any difference in their
properties. This means that a distinction between
self-association of proteins and their complexing may
disappear. In this case, the probability that a given protein
molecule may have in its environment molecules of both the
same and the second protein is 0.5, i.e. the same. The zero
value of the interaction parameter XPRI-PR2 is characteristic
of this situation. Accordingly, as parameters XPRI .S and
XPR2.S approach one another, incompatibility of proteins
should be decreased because of the decrease of both the
Ax-effect and the XPRI.PR2 parameter. This means that the
Ax-effect is of primary importance for thermodynamic
incompatibility of proteins. Apparently, for this reason,
incompatibility is typical of proteins belonging to the
different classes within the Osborne classification, i.e, of
proteins differing in their hydrophilicities or interaction
parameters, XPR-S'
The molecular weight of many oligomeric proteins
contain ing several polypeptide chains is usually dependent
on solvent conditions, particularly on pH (29,30,33-36). A
decrease in the molecular weight (i.e. an increase in the
negentropic effect due to the concentration of the proteins
in the different phases) should result in decreased
incompatibility of proteins.
Unfolding of protein molecules (e.g. in concentrated urea
or guanidinium hydrochloride solutions) should probably
enhance protein incompatibility since lower values of phase
separation thresholds are typical of flexible chain polymers.
As proteins are polyelectrolytes, electrostatic interactions
are a significant factor affecting protein incompatibility. In
concentrated solutions of polyelectrolytes, electrostatic
interactions appear to be largely screened. For this reason,
the electrostatic interactions are confined to a short-range
repulsion between macromolecules (32), which bring a
negative contribution to the interaction parameter XPR.S'
This corresponds to an improvement in solvent quality with
respect to the protein. This contribution could decrease by an
174 V.l Polyakov. V. Ya. Grinberg and V.B.Tolstoguzov

order of magnitude with an increase in salt concentration. By
contrast, this contribution could be increased with an
increase in average charge of a protein molecule.
We will now consider two proteins that differ in accessible
surface hydrophilicity. This difference between proteins
reflects their difference in amount of accessible hydrophilic
groups that are normally ionizable. The parameter XPR-S of
a and incompatibility are related, it can be assumed that the
....9
><
the more hydrophilic protein should depend on pH and salt
concentration to a greater extent than that of the less
hydrophilic protein. Therefore, it can be expected that the
Ax-effect should decrease with an increase in salt
concentration. By contrast, the Ax-effect should increase
with a shift of pH away from the protein isoelectric point (pI)
(Fig. 2). This assumes that the proteins do not differ
significantly in their isoelectric points. Since the Ax-effect
incompatibility should be diminished with an increase in salt

t
III
S
concentration and be enhanced with increasing pH, when the
pH exceeds the protein pI.
~
I
Q.
>< 2 It should be stressed that these assumptions are in
agreement with the experimental data (Fig. 3). Some
decrease in incompatibility within the pH range from 6.0 to
a 1/C~
s
8.0 could be due to a superposition of the effects of casein
dissociation and changes in the Ax-effect. In this pH range,
b the apparent mol. wt of casein decreases from 180 to 50 kDa.

t
III
I
1 It can also be assumed that electrostatic effects play an
additional important role in protein incompatibility (37).
When a protein I-protein 2-water system separates into two
~
Q.
><
-------2
pH~
phases with protein 1 and protein 2 predominating in each
phase, the electrostatic free energy of the system is likely to
decrease. The reason is that the concentration of each protein
is now higher in its phase than it was in the mixed system.
Alternatively, it is known (32) that the higher the
Figure2 Schematic dependences of the protein-solvent interaction polyelectrolyte concentration, the stronger the screening of
parameters upon salt concentration (a) and pH (b) for two proteins electrostatic macro-ion interactions and the lower the
(l,2) differing in the hydrophilicities of accessiblemolecular surfaces, absolute contribution of the electrostatic component in the
where the hydrophilicity of protein 2 is higher than that of protein I. free energy of the system.
LlXo is a value of the ~x-effect at infinitely large salt concentration
It is also of importance that the partial entropy of
where the electrostatic interaction is completely screened. This value
counter-ions, the main component of the mixing entropy of
is only defined by a difference in hydrophilicities of accessible
molecular surfaces of the proteins. The slope of the dependences is
the system, is not significantly changed since the bulk of the
roughly proportional to the square of the average charge of the system is accessible to counter-ions both before and after
proteins. In tum, the average charge is roughly proportional to a phase separation. In other words, a decrease in free energy
number of accessible hydrophilic residues of the protein. For this resulting from demixing of the protein I-protein 2-water
reason, the XPR-S interaction parameter of the more hydrophilic system may be due to an increase in the concentration of
protein 2 depends more strongly on salt concentration. macro-ions and to a more complete screening of their

a b c

0.50

~ 0.25
z

o o

Figure3 Phase diagrams of some protein I-protein 2-water systems at different salt concentrations (a, b) and different pHs (c): (a) PRI :::
gelatin, PR2 =legumin (pH 6.7, t =40°C); (b) PRI =casein, PR2 =soybean globulin fraction (pH 6.9, t = 20°C); (c) PRI =casein, PR2 :::
soybean globulin fraction (total protein concentration is constant at 20%; [NaCl] =0.0, t = 20°C). Regions of incompatibility are shaded.
Apparent splitting of the diagrams in (b) results from the fact that the upper and lower parts were determined at different constant ratios of
concentrations of the proteins ([PRI]/[PR2] = 0.5 and 0.667, respectively).
 Thermodynamic incompatibility ofproteins 175

electrostatic interactions without significant losses of the
mixing entropy. Such a spontaneous concentration of
macro-ions is not possible for a binary protein-water system
since it would be accompanied by an increase in the
concentration of counter-ions and, consequently, a large
negentropic effect. Evidently, the considered situation will
change slightly in the case of a mixture of proteins differing
substantially in their charges. Then, the counter-ions may be
partitioned non-uniformly between co-existing phases. The
counter-ion concentration in the phase of the protein with
larger charge seems to be higher. This should certainly be
associated with some negentropic effect at the phase
separation. However, at the same time, it needs to be noted
that in practice it is rather difficult to find two food proteins
contrasting in their isoelectric points and hence having rather
different charges at the same pH . Thus, the noted
negentropic effect is not apparently of primary importance
for food proteins.
Some features of phase equilibria in the protein
I-protein 2-water systems
Phase diagrams for some protein I-protein 2-water systems
are presented in Figures 4 and 5. Normally, they are strongly
asymmetrical. The critical point does not usually coincide
with the phase separation threshold and the binodal curve is
located close to one of the concentration axes. A divergence
of the critical point and the separation threshold reflects
the ~x-effect (38). The critical point is displaced from the
separation threshold towards the axis of the protein with the
lower hydrophilicity and, therefore, the larger interaction
parameter XPR -S' This displacement may be so large that the
critical point falls outside the experimentally accessible area
of system composition.
When the distance between the critical point and the phase
separation threshold is used as a measure of the ~x-effect,
the proteins studied form the following series of decreasing
interaction parameter XPR-S: gliadin> soybean (SB) globulin
fraction> 'casein > ovalbumin> gelatin. The hydrophilicity
of proteins by Osborne increases in the order: prolamins <
globulins < albumins.
There is obviously a correlation between these two series.
This means that the Osborne classification can be used for
predicting protein components that will be mainly
concentrated by phase separation of a protein I-protein 2-
water system.
It should be noted that there is a reverse qualitative
correlation between the phase separation threshold and the
distance between the critical point and the phase separation
threshold. The phase separation threshold also directly

a b c
25 25 25

20 20 20
?R ?R ?R

z 15 z15 Z 15
w w w
(/)
« 10
(J)
~ 10 « 10
u u u
5 5 5

0 0
0 5 10 15 20 25 0 5 10 15 20 25 5 10 15 20 25
OVALBUMIN. % SB GLOBULIN FRACTION % GLIADIN %

?R d
:i 25
0
i= 20
u : ) - 1
-c
e: 15 • - 2
z
::::i 10
::::l
en
0
.--J
5
-
~ - 3
-
4

0
m 0
(f) 0 5 10 15 20 25
OVALBUM IN ,%

Figure 4 Phase diagrams of some protein I-protein 2-water systems at 20°C: I, binodal; 2, critical point; 3, separation threshold; 4, region
of possible location of the critical point. Regions of incompatibility are shaded. The separation threshold in percent is given in the upper
right-hand corner of each diagram . The pH of system is 6.6 (a, d), 6.9(b) and 11 .0 (c). The app arent mol. wt is 100 kDa (a) and 30 kDa (c) for
casein, 45 kDa for ovalbumin, 290 kDa for soybean globulin fraction and 20 kDa for gliadin . Proteins are presented according to the Osborne
classification in order of decreasing hydrophilicity: albumins (a, d); globulins (b); prolamins (c). The critical points presented in this and other
phase diagrams were found as intercept points of binodals and rectilinear diameters determined by the phase volume ratio method (19).
176 V.I. Polyako v, V. Ya. Grinberg and V. B.Tolstoguzov

a b
~ 25 ~ 25
z z
0 0
t- 20 t- 20
0 0
-c <:
c:::
c::: 15 15
u, u,

Z Z
.-J 10 .-J 10
~ ~
co co
0 5 0 5
.-J .-J
c.:> 0
co 0 co
(IJ
Cfl 0 5 10 15 20 25 5 10 15 20 25
0 - 1
GELATIN , %
CASEIN, %
c • -- 2
3 d
25
" 25

20 20
~
~

z
15 z - 15
t-
i=
<:
-c
.-J
.-J 10 W
10
W c.:>
c.:>
5 5

0
0 5 10 15 20 25 5 10 15 20 25
BB GLOBULIN FRACTION % LEGUMIN , %

Figure 5 Phase diagrams of some protein I-protein 2-water systems (continued): 1, binodal; 2, critical point; 3, separation threshold.
Regions of incompatibilityare shaded. The separation threshold in percentis given in the upper right-hand comer of each diagram. The pH
and temperature of systemare 6.9 and 20°C (a); 7.0 and 40°C (b-d). The apparent mol. w t is 290 kDa for soybean globulin fraction, 100 kDa
forcasein, 280 kDa for broad bean globulin fraction, 160 kDa forgelatin and 350 kDa for legumin. Casein, legumin and the globulin fractions
have a globular conformation, and gelatin has a random coil conformation.

reflects the magnitude of the Ilx-effect. The larger the
Ilx-effect, the lower the separation threshold and accordingly
the stronger the incompatibility. This result displays an
important role of the Ilx-effect in protein incompatibility.
The second type of asymmetry of phase diagrams of
protein I-protein 2-water systems is an inequality of the
asymptotes of binodals at high protein concentrations. This
may correspond to a difference in molecular weight between
proteins. The binodal is always located close r to th e
concentration axis of the protein with the lower molecular
weigh t. Th is feature is one of the most typical of phase
diagrams for binary mi xtures of components differing
strongl y in molecular weight (Fig. 6). According to van der
Waals (39), the critical point of such a system should be
di splaced to the axis of the component with the lower
molecular weight. The Flory-Huggins theory gives the
mathematical expression for this (40):
( I)
where lj>c1 and lj>c2 are the critical volume fraction s of
components of the polymer 1- polymer 2 system; M], M 2
and VI, V2 are molecular weights and specific partial volumes
of these components.
It may be assumed (Fig. 6a) that the separation threshold
of the polymer I-polymer 2-solvent system at T-constant
lies in a plane that passes through the critical point of the
polymer I-polymer 2 system and the solvent apex S of the
Gibbs triangle, i.e. on the straight line connecting a
projection of the critical point (A,A') on a T-constant plane
with th e apex S. Each point along this line represents systems
with a constant concentration ratio of polymer I and
polymer 2. As specific partial volumes of proteins do not
differ significantly (4 1), the following relationship between
protein concentrations (C *PR]' C*PR2), corresponding to the
separa tion thre shold of a protein I-protein 2-water system,
and molecular weights (M], M 2) of these proteins would be
expected:
(2)
This relationship is in quite satisfactory agreement with the
experimental data (Fi g. 6b). Thus, the asymmetry of a
location of the separat ion threshold in a protein I-prot ein
2-water system is probably due to a difference in mole cular
weight between the proteins. A mixed solutio n
corresponding to the phase separation threshold is not able
for having a higher concentration of the protein of lower
molecular weight.
Figure 5 illustrates the effects of the conformational state
 Thermodynamic incompatibility ofproteins 177

a b
s
5

4
OJ
a:
..o Q.
3
--a:
.. Q.
2
o

2
A A'

Figure 6 Plots illustrating the effect of varying the ratio of polymer molecular weights on the position of the separation threshold. (a)
=
Isothermal section of a phase diagram of some polymer I-polymer 2-solvent (S) systems: 1, binodal at M) M2; 2, binodal at M) < M2; 3,
separation threshold; 4, projection of the critical point of the system (scheme). SA and SA' are lines of a constant ratio of weight polymer
concentrations. (b) Dependence of the ratio of weight concentrations of the proteins, corresponding to separation thresholds of the protein
I-protein 2-water systems under investigation, on the ratio of the molecular weights of proteins. Straight line is consistent with the theoretical
dependence (2).

a b
30 30

~
25 L ~
25 ~
z 20 z 20
~ ~
::::> 15 ::::> 15
CD rn
.J .J
« 10 <t:. 10
> >
0
0
5 5

0 0
0 5 10 15 20 25 30 0 1 0 5 10 15 20 25 30
OVALBUMIN % •--
2
3
BSA, %

<l\! 30
c "
.- 4 ~ 30
d

CIl
a::: 25 vi 25
w a:::
l-
w
I-
CIl 20 (f) 20
::::> ::::>
.J .J
u 15 u 15
0 0
~ ~
a:::
w
10 a::: 10
w
I I
l- 5 I- 5
<t:. <t:.
0 0 0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
OVALBUM IN, % BSA , %

Figure 7 Phase diagrams of some protein I-protein 2-water systems at pH 7.6 and a temperature of 20°C before (a, b) and after (c, d) thermal
denaturation of one of the proteins (namely, ovalbumin): 1, binodal; 2, critical point; 3, separation threshold; 4, region of possible location of
the critical point. Regions of incompatibility are shaded. A separation threshold in percent is given in the upper right hand-corner of each
phase diagram . Ovalbumin (OA) thermo-clusters were obtained by heating 5% solutions of the protein at 100°C for 1 h. The apparent mol. wt
is 45 kDa for ovalbumin, 69 kDa for BSA and 760 kDa for the ovalbumin thermo-clusters.

and of molecular weight of proteins on incompatibility. This gelatin-water (b). Gelatin has a random coil conformation
figure shows phase diagrams for two systems: SB globulin that is more unfolded compared to that of casein.
fraction-easein-water (a) and the SB globulin fraction- Accordingly, the concentration region corresponding to
178 V. I Polyakov, V. Ya.Grinberg and V.B. Tolstoguzov
two-phase systems (i.e. the incompatibility region) is larger
for the system containing gelatin than for the system
containing casein. Consequently, unfolding of protein
molecules is favourable for incompatibility with other
proteins.
Two other systems, the broad bean (BB) globulin
fraction-gelatin-water (c) and legumin-gelatin-water (d),
are mainly notable for the difference in molecular weight
between the two preparations of the seed storage globulins
and between them and gelatin. The difference between the
BB globulin fraction (~280 kDa) and legumin (350 kDa) (42)
results in the latter system having a lower phase separation
threshold. Also, the binodal curve of this system is closer to
the concentration axis of gelatin, i.e, the protein of lower
molecular weight.
As mentioned above, protein incompatibility is quite a
conservative phenomenon, only slightly affected by factors
such as pH and ionic strength. Figure 7 shows the possibility
of controlling the compatibility of proteins by thermal
denaturation. Thermal denaturation of a protein at the
concentration lower than the critical concentration for
protein gelation can lead to the formation of protein clusters
of controllable size, shape and hydrophilicity (43). Figure 7
shows that ovalbumin is completely compatible with
BSA. However, the thermo-clusters of ovalbumin are
incompatible with both BSA and native ovalbumin. A
control of denaturation conditions, including the
composition of the solvent used, allows protein clusters
differing in molecular weight and hydrophilicity to be
prepared. This, in turn, allows the phase state as well as the
composition and rheological properties of co-existing phases
of protein-containing food systems to be controlled. This
method of governing the structural functions of food
proteins is of great applied importance. For instance, it is of
interest for processing two-phase protein I-protein 2-water
systems into fibrous texturates by spinneretless spinning
(44,45).
Conclusion
Proteins of different classes, according to the Osborne
classification, are incompatible in aqueous media. Owing to
compactness of the protein molecules, phase separation
normally takes place only at high protein concentrations. The
most important factor governing protein incompatibility is
the difference in hydrophilicity between proteins, i.e. the
!lx-effect: the larger the difference in hydrophilicity of the
proteins, the more pronounced their incompatibility.
Electrostatic interactions are also of primary importance
in protein incompatibility. Electrostatic interactions can
contribute to a decrease in electrostatic free energy resulting
from phase separation of a protein I-protein 2-water
system. Accordingly, protein incompatibility is diminished
with an increase in ionic strength and enhanced with an
increase in net charge of the protein molecules.
The asymmetry of phase diagrams for protein I-protein
2-water systems depends on the molecular weight ratio of
the proteins. The binodal curve is always located closer to the
concentration axis of the protein of lower molecular weight.
Thermal denaturation of proteins (at concentrations
below the critical concentration for protein gelation) is a
promising technique for enhancing incompatibility of
proteins. This can be of importance for protein functionality
in both traditional and novel foods.
Acknowledgements
The authors are grateful to Mrs Elizabeth Prior for editing
the manuscript.
References
1. Tolstoguzov,v. (1988) Food Hydro coIl., 2,339-370.
2. Tolstoguzov,v. (1991) Food Hydrocoll. , 4, 429-468.
3. Tolstoguzov,V. (1992) In Phillips,G.O., Williams,P.A. and
Wedlock,D.1 (eds), Gums and Stabilisers for the Food
Industry. IRL Press, Oxford, Vol. 6, pp. 241 -266.
4. Tolstoguzov,v. (1993) In Dickinson,E. and Walstra ,P.
(eds), Food Colloids and Polymers: Stability and
Mechanical Propert ies. Special Publication No. 113,
Royal Society of Chemistry, Cambridge, pp. 94-102.
5. Tolstoguzov,v. (1994) In Nishinari ,K. and Doi,E. (eds),
Food Hydrocolloids; Structure, Properties and Functions.
Plenum Press, New York, pp. 327-340.
6. Tolstoguzov,v. (1994) In Phillips,G.O., Wiliiams,P.A. and
Wedlock,D.1 (eds), Gums and Stabilisers fo r the Food
Industry. Oxford University Press, Oxford , Vol. 7, pp.
115-1246.
7. Scatchard,G., Gee,A. and Weeks,J. (1954) J Phys. Chem .,
58, 783-787.
8. Scatchard,G. and Pigliacampi.I (1962) JAm. Chem.
Soc.,84,127-134.
9. Ogston,A.G. (1937) Biochem. J, 31,1952-1957.
10. Ogston,A.G. (1962) Arch. Biochem. Biophys., 1(Suppl.),
39-51.
11. Edmond,E. and Ogston,A.G. (1968) Bio chem . J , 109,
569-576.
12. Braudo,E.E. and Tolstoguzov.VB. (1974) Nahrung, 18,
173-184.
13. Antonov,Yu .A., Grinberg,V.Ya. and Tolstoguzov,V.B.
(1979)Nahrung,23,207-214.
14. Cohn,E.1 and Edsall,lT. (1943) Proteins, Amino A cids
and Peptides as Ions and Dipolar Ion s. Reinhold
Publishing Corp., New York, pp. 569-585.
15. Czok ,R. and Bucher,T. (1960) Adv. Protein Chem ., 15,
315-415.
16. Dixon,M. and Webb,E.C. (1961) Adv. Protein Chem., 16,
197-219.
17. von Hippel,P.H. and Schleich,Th. (1973) In Timasheff,
S.M. and Fasman,G.D. (eds), Structure and Stability of
Biological Macromolecules. Mir, Moscow, pp. 320-480 (in
Russian).
 Thermodynamic incompatibility of proteins 179

18. Papkov,S.P. (1981) Phase Equilibria in Polymer-Solvent M itchell, lR . (eds) , Food Structure-Its Creati on and
Systems. Khirnia, Moscow (in Russ ian) . E valuation. Butterworths, London, pp. 181-196.
19. Polyakov,Y.L, Grinberg,Y.Ya., Antonov.Yu.A, and 45. Tolstoguzov.V (1993) J Am. Oil Chern. Soc., 70, 417--424.
Tolstoguzov,Y.B. (1979) Polym. Bull., 1,593-597. 46. Polyakov.Vl. (1987) The thermodynamic com p atibility
20. Polyakov,V.L, Grinberg.VYa. and Tolstoguzov.VB. of proteins in solutions. PhD Thesis, Institute of
(1980) Polym . Bull., 2, 757-760. Organo-Element Compounds, USSR Ac ademy of
21. Polyakov.Vl ., Kireyeva ,O.K. , Grinberg,Y.Ya. and Sciences, Moscow (in Russian).
Tols toguzov,Y.B. (1985) Nahrung , 29, 153-160.
22. Polyakov.VL , Popello , LA. , Grinberg,Y.Ya. and Received on January 5, 1996; accepted on May 10, 1996
Tolstoguzov.VB. (1985) Nahrung , 29,323-333.
23. Andersso n,O., Schmandke.,H., Polyakov,Y.L, Grinberg,
Y.Ya., Bikbov,T.M ., Danilenko,A .N., Leontjev,A.L. and Appendix
Tolstoguzov,Y.B. (1985) J Food sa, 50, 1133-1136.
24. Polyakov.VL, Popello,LA., Grinberg,Y.Ya . and
Tolstoguzov,Y.B. (1986) Nahrung , 30, 81-88. Table Al Binodal curve for ovalbumin (1)-8B globulin fraction
25. Pol yakov,Y.L, Popello,LA. , Grinberg,Y.Ya. and (2)-water system at pH 6.6 and t = 20°C (20)
Tolstoguzov,Y.B. (1986) Nahrung , 30,365-368.
No. Cl, % C2,% No. C l, % C2, %
26. Osborne,T B. (1924) Vegetable Proteins. Longmans,
Green, New York . 1 18.4 0.0 5 9.6 10.0
27. De G ennes,P. (1982) S caling Concepts in Polymer Phys ics. 2 15.3 0.6 6 9.0 12.3
Mir, Moscow, pp, 105-1 41 (in Russian). 3 12.2 3.9 7 8.2 17.2
28. Tanford,C. (1965) Physical Chemistry of Macromolecules. 4 lI.l 6.2 8
Khimia , Moscow, pp, 367-517 (in Russian).
29. Tanford,C. (1968) Adv. Protein Chern., 23,121-282 ;
30. Tanford,C. (1970) Adv: Protein Chern. , 24, 1-95 .
31. Shulz.G. and Schirmer,R. (1982) Principles of Protein TableA2 Binodal curve for gelatin (I )-BB globulin fraction
Structure. Mir, Moscow (in Russian). (2)-water system at pH 7.0 and t = 40°C (22)
32. Khokhlov,A.R. and Khachaturian,K.A. (1982) Polymer,
No. Cl , % C2,% No. Cl, % C2,%
23, 1742-1750.
33. Lapanje.S. (1978 ) Physicochemical Aspects of Protein I 16.2 0.9 5 1.7 15.1
Denatu ration. Wiley Interscience., New York . 2 14.4 0.9 6 1.4 17.9
34. Klotz,LM ., Darnall,D.W and Langerman,N.R . (1975) In 3 10.4 0.9 7 1.2 20.8
N eurath,H . and HiII,R .L. (eds), The Proteins. Academic 4 9.4 1.0 8
Press, New York, Vol. I, pp. 293-411.
35. Wolf,Wl (1972) In Smith,A.K. and Circle,SJ. (eds),
Soyb eans. Chem istry and Technology. Prote ins. AVI
Publishing Co . Inc. , Westport, Vol. I, pp. 93-143. Table A3 Binodal curve for gelatin ( I)-legumin (2)-water system at
36. Mckenzie,H .A . (1967) Adv. Protein Chern., 22, 55-65. pH 7.0 and t = 40°C (22)
37. Vasilevskaya.V'V , Starodubzev,S.G. and Khokhlov,A.R.
No. Cl,% C2,% No. Cl, % C2,%
( 1987) Vysokomolek. Soed., 29B , 930-933 (in Russian ).
38. Grinberg,Y.Ya. and Tolstoguzov,V.B. (1997) Food 1 16.1 0.0 5 0.8 23.1
Hydrocoll., th is issue. 2 7.3 0.4 6
39. van de r Waals,lD. and Kohnstamm,Ph. (1912) Lehrb. 3 3.6 6.7 7
Thermodyn. Leipz ig, II , S. 477 . 4 0.8 22.0 8
40. Tompa .H . (1956) Polymer Solutions. Butterworths,
London.
41. Cohn,E.J. and Ed sall ,lT. (1943) Proteins, Amino Acids
and Peptides as Ions and Dipolar Ions. Reinhold Table A4 Binodal curve for gelatin (I )-legumin (2)-water system at
Publishing Corp., N ew York , pp. 370-381. pH 7.0, 0.5 mol/drrr' NaCl and t = 40°C (22)
42. Derbish ire,E ., Wright,D.l and Boulter,D. (1976) No. Cl , % C2,% No. Cl,% C2,%
Phytochemistry , 15, 3-24.
1 12.6 4.3 5 2.8 18.7
43. Grinberg,Y.Ya., Grinberg,N.Y., Bikbov,TM.,
2 8.1 4.9 6 2.2 20.6
Bronich,TK. and Mashkevich,A .Ya. (1992) Food
3 5.7 6.6 7
Hydro coll., 6, 69-96.
4 4.1 13.6 8
44. Tolst og uzov,V.B. (1988) In Bla nsh ard,IM.Y. a nd
180 V.l Polyakov, V. Ya. Grinberg and V.B.Tolstoguzov

TableA5 Binodal curve for ovalbumin (Ij-casein (2)-water system at TableA7 Binodal curve for casein (1 )-gliadin (2)-water system at
pH 6.6 and t = 20°C (20) pH 11.0 and t = 20°C (20)

No. Cl.% Cz,% No. Cl,% Cz,% No. Cl.% Cz,% No. Cl,% Cz,%
1 24.0 0.0 5 12.8 8.8 1 11.9 1.3 5 3.0 2.0
2 17.6 3.6 6 12.1 9.8 2 9.8 1.3 6 0.0 6.0
3 16.0 3.6 7 11.1 11.5 3 7.3 1.4 7 0.0 7.9
4 15.0 4.8 8 9.6 15.7 4 3.8 1.8 8

TableA8 Binodal curve for BSA (I)-OA TCa (2)-water system at pH

6.7 and t = 20°C (24)
No. Cl.% Cz,% No. Cl,% Cz,%
1 29.9 0.4 5 15.7 3.6
TableA6 Binodal curve for casein (I)-SB globulin fraction (2)-water 2 29.4 0.4 6 12.2 5.0
system at pH 6.9 and t = 20°C (19,20) 3 28.8 0.4 7 10.2 6.3
No. Cl,% Cz,% No. Cl,% Cz,% 4 26.2 0.5 8 9.3 7.3

1 14.0 0.3 9 8.1 5.3 aThermo-custers of ovalbumin prepared by heating 5% ovalbumin

2 13.3 0.2 10 7.3 6.8
e
aqueous solution with pH 6.7 at 1000 for 1 h followed by a
lyophilization.
3 13.1 0.2 11 6.6 9.6
4 13.0 0.3 12 6.3 10.2
5 9.8 1,9 13 6.2 10.6
TableA9 Binodal curve for ovalbumin (1 )-OA TC (2)-water system
6 9.9 2,2 14 6.0 11.5 at pH 6.7 and t = 20°C (24)
7 9.0 3.1 15 5.1 14.4
8 9.0 3.9 16 5.5 15.3 No. Cl,% Cz,% No. CI,% Cz,%
30.0 0.4 5 8.8 5.2
24.8 0.4 6 7.9 7.3
3 18.6 0.4 7
4 10.8 2.5 8

TableAlO Critical points and phase separation thresholds of some protein l-protein 2-water systems (44)
No. Protein 1 Protein 2 Critical point Separation threshold Conditions
Cl,% Cz,% Cl,% Cz,%
Ovalbumin SB globulin <10 >15 12.1 3.9 pH 6.6, 20°C
fraction
2 Gelatin BB globulin 2.5 11.6 5.0 3.8 pH 7.0, 40°C
fraction
3 Gelatin Legumin 2.1 12.0 5.0 3.4 pH 7.0, 40°C
4 Gelatin Legumin 4.0 16.0 8.0 5.0 pH 6.6, 40°C, 0.5
moVdm3NaCI
5 Ovalbumin Casein 13.9 6.5 15.9 3.8 pH 6.6, 20°C
6 Casein SB globulin 7.0 8.0 9.0 3.0 pH 6.9, 20°C
fraction
7 Casein Gliaditl <5 >10 3.0 2.5 pH 11.0, 20°C
8 BSA OATca <10 >15 12.2 5.0 pH 6.7, 20°C
9 Ovalbumin OATC· 10.4 3.6 10.8 2.5 pH 6.7, 20°C
aThermo-c1usters of ovalbumin prepared by heating 5% ovalbumin aqueous solution with pH 6.7 at 1000e for 1 h followed by a
lyophilization.