ELS EVI E R Current Opinion in Colloid & Interface Science 5 (2000) 188-194

www.elsevier.nl/locate/cocis

Globular protein gelation

Walraj S. Gosal, Simon B. Ross-Murphy"

Biopolymers Group, Division of Life Sciences, King's College London, Franklin- WilkinsBuilding, 150 Stamford Street,
London SEl8WA, UK

Abstract

In this paper, significant advances in the structure and rheological properties of globular protein gels have been described.
The achievements of scattering methods, which have provided information over a range of distances from the monomer to the
micron scale, are reviewed. The kinetics of gelation measured by rheological methods have also been discussed, including
currently applied models for the gelation time, the gel elastic modulus and the critical gel concentration. 0 2000 Elsevier
Science Ltd. All rights reserved.

Keywords: Gel; Gelatine; Rheology; Protein denaturation; Peptide networks; Amyloid

1. Introduction For example, it is now appreciated that in simple

Some aspects of heat-set gelation of globular pro-
tein solutions are familiar to anyone who has boiled
an egg. Nevertheless, a detailed understanding of
factors controlling the rate of gelation and the final
'gel strength' achieved and how these depend on the
implicit variables, such as the protein sequence, con-
centration, pH and ionic strength, are less well es-
tablished. Historically, the picture outlined in Ferry's
review of 1948 [lo] is that on heat-induced denatura-
tion, a protein completely unfolds, and then inter-
molecular interactions lead to the formation of a
fine-meshed 'macromo1ecular' gel. This principle is
still quite appropriate for gelatine [21 and for some
chemically denatured globular proteins, but for heat-
set globular proteins the picture is rather different.
The article published in this series in 1996 by Clark
serves as an invaluable introduction [3'], as does his
updated monograph contribution [4"].
* Corresponding author. Tel.: + 44-20-7848-4081; fax: + 44-20-
7848-4500.
E-mail address: simon.ross-murphy@kcl.ac.uk (S.B. Ross-
Murphy).
heat-induced denaturation, the protein size and shape
is only mildly perturbed. Instead, some of the hy-
drophobic groups, which at ambient temperatures re-
main buried in the protein core, become exposed
above some minimum unfolding or denaturation tem-
perature, T,. (Here, we have distinguished between
this value corresponding to the start of a DSC en-
dotherm, and T,, the temperature at the DSC maxi-
mum.) This 'hydrophobic effect' leads to aggregation
to form either fine stranded networks, or amorphous
particulate structures of a 'physical gel' [5]. At sec-
ondary structure level this usually means that there is
a small decrease in the a-helix component, and an
increase in the proportion of the P-sheet [4"], and
the new technique of attenuated total reflection
Fourier transform IR (ATR FT-IR) has been em-
ployed for P-lactoglobulin systems @-Lg) [61.
Unfortunately efforts to relate secondary structural
changes to long-range structure and rheology have
been relatively unsuccessful. Complications are due to
a number of additional factors, and the precise balance
of pre-gel intermediates appears controversial [7,8"].
The problem is, firstly, that disulfide (i.e. chemical)
cross-links can contribute to the gel structure, and

1359-0294/00/$ - see front matter 0 2000 Elsevier Science Ltd. All rights reserved.
PII: S 1 3 5 9 - 0 2 9 4 ( 0 0 ) 0 0 0 5 7 - 1
 WS.Gosal, S.B. Ross-Murphy /Current Opinion in Colloid & Integace Science 5 (2000)188-194
their presence is introduced preferentially at some pH
values [9]; secondly, there appears to be a pre-aggre-
gation step involving the formation of dimers and
higher oligomers [lo']. At pHs around the protein
isoelectric point, PI, and high extrinsic ionic strengths,
the resultant gel networks are heterogeneous and
largely opaque, and even show some syneresis, just as
we would expect for an 'egg-white' gel. At lower levels
of added salt, much more transparent gels are formed
[ll"]. Finally, under other conditions, for example at
pH values well below the isoelectric PI, fine but highly
persistent fibrillar networks are formed rather than
particulate stranded gels [12] and cold-set gels are
formed at pH 3.5 [13]. Such rod-like structures appar-
ently form by side-to-side aggregation, and their per-
sistence length varies inversely with ionic strength
[14"1. They are of particular interest, however, be-
cause of the apparent parallels with p-amyloid fibrils
[15"]. Amyloid is a term that describes an abnormal
deposition of dense and insoluble fibrillar protein,
which damages organs or tissues, especially the brain.
Amyloid deposits occur both in the so-called prion
diseases, such as BSE and new variant CJD, and
dementias such as Alzheimer's and Huntington's
chorea [16'].
The present article will, because of required brevity,
concentrate largely on longer distances macromolecu-
lar probes, such as small angle neutron and X-ray
scattering, and rheological measurements. This ne-
glects the considerable literature on multifarious
spectroscopic and microscopic techniques. It will also
concern itself mainly with measurements on single
component heat-set protein systems, although some
work on protein mixed gels is included [17-191. Nev-
ertheless this ignores the very extensive work on whey
protein mixtures, much of it published in food and
dairying journals, where structure-property relation-
ships are very difficult to establish. Furthermore, al-
though presumably all globular proteins above the
critical gelation concentration, C,, can gel given ap-
propriate conditions, we have concentrated in this
work on bovine serum albumin (BSA) and p-lacto-
globulin (p-Lg) gels. This is because the vast majority
of work has been on these, with relatively few papers
on, for example, ovalbumin, soy proteins and other
whey proteins, such as a-lactalbumin and immuno-
globulins.
2. Peptide assembly
Although most of the papers concern proteins per
se, some of the most interesting recent work has
concentrated on developing model systems using pep-
tides. Such networks will form if thermodynamics
favours ordered but soluble 'superstructures'. Both
189
the motivation for assembly and the solvent affinity
may arise either from the peptide sequence or be
tailored synthetically [15",20,21'].
p-Sheet fibrils have received the most attention.
Here assembly arises from favourable solvation ener-
gies and side chain interactions accompanying the
formation of p-sheet structures. The latter can be
constructed using short sequences of alternating hy-
drophobic and charged residues [22]. Increasing the
hydrophobicity of the side chains increases the
strength and the self-assembling capacity [15",22],
whereas charge and diverse recognition mechanisms
dictate the arrangement or morphology of the net-
work [21',23"].
Boden et al. were able to design anti-parallel p-
sheet 'tapes', by restricting to one dimensional assem-
bly. These formed highly entangled gel-like systems,
and visco-elastic properties could be dictated by ei-
ther shear history or chemistry; the latter by altering
solvent polarity or H-bonding ability. Both had dra-
matic effects on p-sheet formation and/or solubility.
Altering the response to such influences could be
achieved by modifying the properties of the p-sheet.
They also synthesised a peptide (DNl), which was
soluble in increasingly polar solvents [24"]. DN1
assumed a random configuration below a critical tape
concentration, above which the p-sheet motif was
formed and assembly took place, indicating nucleated
as opposed to spontaneous self-assembly. A distinc-
tion between these remains elusive.
The peptide repeat sequence (VT),, has been ex-
ploited in two cases. Janek et al. [25] found that a
minimum of 12 residues were required to form stable
p-sheet fibrils, while Lashuel et al. [21'] were able to
employ only eight residues, by interposing a bulky
aromatic group. A vast array of fibrillar structures
(protofilaments, fibrils and ribbons) could be obtained
simply by changing the pH, or ionic conditions, which
modified the rate of assembly. An extreme range of
morphologies, including hollow tubes, distorted and
branched fibrils could be achieved by using peptides
incorporated into amphiphilic lipids [26].
Another useful motif is the coiled-coil [20,27]. Both
pH and temperature can control (dis)assembly, mak-
ing the resultant structures thermoreversible, and the
response [20,27] can be encoded into the peptide
sequence. Such systems have potential applications as
molecular switches [20]. Finally, conformational
switching from a coiled-coil to p-sheet fibril has been
achieved by introducing acyl chains [15"]. This intro-
duces a hydrophobic defect, the extent of which gov-
erns assembly.
A related area is the study of protein structure and
unfolding in non-aqueous solvents [28-311 or by
high-pressure [32-351 in order to probe structural
features. Such model peptide and protein studies are
190 W S . Gosal, S.B. Ross-Murphy /Current Opinion in Colloid & Interface Science 5 (2000)188-194

clearly increasing, and may prove invaluable in future

years.
B /
3. Scattering measurements

Early scattering work was, perhaps naturally, car-

ried out using conventional laboratory X-ray genera-
tors, but the availability of neutron and synchrotron
sources over the last 15 years or so, has completely
revolutionised the area, because the high intensity (I)
allows real-time experiments to be carried out. The
principles of scattering are presumed to be familiar to
most readers, in that the size probed is inversely
proportional to the scattering vector, q, which de-
pends upon the electromagnetic radiation wavelength
and the scattering angle. Consequently wide-angle
X-ray scattering (WAXS) explores individual proteins,
-
down to say 0.2 nm resolution, whereas small angle
(SA)XS, or, employing neutrons, S A N S , can measure
aggregation stages from the monomer up to, say,
1000-mers. Much work has concentrated on determin-
ing monomer-dimer ratios at different pHs and ionic
strengths [10',34,36-38',39-431. Summarising the
overall conclusions is not easy because of the very
different conditions employed, but Fig. 1 illustrates Fig. 1. Formation of heat set gels: model representing the possible
the hierarchy of aggregation steps. aggregation steps in a typical heat-set globular protein such as
After extended heating times, small-angle scatter- p-Lg. Both dimer-monomer and monomer to denatured monomer
ing results usually show an almost power-law depen- equilibria are shown. At pH values well below the isoelectric PI
dence of log I ( q ) vs. log q. The extent and precise fibrils are formed simply from aggregated monomers (A). Under
other conditions, (B), a pre-aggregate is formed, which in turn leads
slope of this power law region is, however, dependent to a more particulate gel. NB: the length scale for the individual
on the conditions. For BSA, under 'isoelectric' condi- pre-gel aggregate 'units' is different for cases A and B.
tions a slope of -4 was found. Under other condi-
tions, lower slopes ( - 2) were seen, which were con-
sistent with a self-similar, fractal structure the presence of NaCl[38']. This suggests that studies
[8",14",44], with dimensions, df,say 1.5-2.5. With on dialysed systems at low pH would be of interest.
such scattering methods, it is certainly possible to
measure a power-law region extending over 3-4

of -
decades of q, but the upper limit corresponds to sizes
1 km.
For colloidal systems, the fractal description, is of
4. Gel rheology and gelation kinetics

course, now very well known, and the description of
particulate aggregates formed by either, diffusion-
limited or reaction-limited cluster aggregation, DLCA
and RLCA, respectively, is well established [451. How-
ever, the gel state reflects the existence of a mega-
molecule, which spans the size of its container, so 1
krn is still a factor > l o 4 smaller. It would appear
impossible to establish structure over the entire range
of relevant distance scales.
By the same token, measurement, say, of
monomer-dimer ratios, [36] while an interesting pur-
suit, has little to contribute to gelation studies
[39,41,43,46]. Nevertheless, recent fluorescence stud-
ies suggest that the dimerisation of p-Lg is induced by
Certain principles of gelation kinetics are universal,
and others are certainly not - indeed many readers
may have encountered papers (not identified here!) in
physics journals where too great a reliance on univer-
sality has been given without appropriate considera-
tion of the elementary aspects of biopolymer sample
handling. For the present systems, Dickinson et al.
[47',48'] have used Brownian dynamics to simulate a
system of aggregating particles.
As far as the experiment is concerned, it is usual to
place a solution on an oscillatory rheometer and heat,
say at a constant rate of 1°C per minute. In this, the
real and imaginary parts of the complex shear modu-
lus, G' and G", can be followed as a function of time
 W S . Gosal, S.B. Ross-Murphy /Current Opinion in Colloid & Integace Science 5 (2000)188-194
I +
Asymptotic G
Fig. 2. Gelation kinetics: concentration and time dependence of the
gel modulus illustrating one simple method for estimating the
gelation time.
(and temperature) [3',49]. The trace observed shows a
characteristic shape for the 'cure' curve of log (G',
G") against time. For the fluid state G" will normally
be greater than G', although for protein solutions,
this is not always the case. On heating, there is an
initial lag time. Then both GI' and G' begin to
increase, provided the concentration ( C ) of protein is
greater than the critical value C,, with G' increasing
faster than G" before both level off. As the concen-
tration of polymer increases, the value of G' at long
(actually asymptotic) times (strictly the equilibrium
modulus G) also increases, while the gelation time,
here identified simply as the time when G' shows its
initial fast increase, decreases (Fig. 2) [50-521. When
-
C > 5C,, G' a C", with n = 2 f 0.25. Measure-
ments made closer to C , give higher values of n, as
indeed they might be expected to do, since for gela-
tion to occur, the equilibrium modulus must show a
discontinuity in the log of the equilibrium modulus,
G, from zero to a finite value. Consequently n must
+. 00 as C +. C,. Some workers have failed to appreci-
ate this point and, as we shall see below, have inter-
preted the power law exponent n at higher concentra-
tions, in terms of a fractal model [53',54,55'].
Overall, remarkably few papers of interest have
been published in the relevant time-window. This is
certainly not because of the interest and importance
of the area, but may reflect the problems in obtaining
reliable data. The first is the precise technique em-
ployed to determine the gelation time. It is now
recognised that analysis of the frequency dependence
mechanical spectrum of a gelling system (the trace of
G' and G" vs. log 01 can, in principle, lead to a
phenomenologically precise description of the gel
point [34]. For chemically cross-linked systems it is
quite possible to determine the time t, (and/or
chemical conversion) when a sol is just converted to a
gel. For physical gels, things are less straightforward.
Since, say, a BSA gel is formed when a -
10% w/w
system is heated, there is very little pre-gel signal to
191
be seen in the rheometer (the pre-gel relative vis-
-
cosity is 2). (As we have mentioned above, at very
small deformations there is evidence that G' > G"
even here.) Unfortunately this is seen at signal levels
quite close to the limit for current instruments, and
any attempt to scan frequencies at the required low
strains is unlikely to be useful. Consequently, we are
left with more ad hoc methods, such as determining
the required discontinuity by extrapolation [501. Hav-
ing done this, however, extremely high power laws
( > 5) can be seen for the dependence of log (l/t,) vs.
log(C). Such exponents are not to be expected on the
basis of simple models, and may reflect elements of
the aggregation process, perhaps proceeding via more
complex kinetics involving both pre-aggregation events
such as denaturation, and co-operative (non-random)
pro-
cesses combining elements of nucleation and growth.
These are observed for p-Lg gels at acid pH values,
where the highly amyloid-like fibrillar gels are seen
[14" I.
Problems also exist employing a heating ramp,
rather than an isothermal jump to the measurement
temperature, since it is not viable to deconvolute
kinetic and equilibrium processes. However, the form
of the final gelation temperature vs. concentration
profile bears a qualitative resemblance to a lower
critical solution temperature (LCST) phase diagram
(Fig. 3) [50]. Other 'phase diagrams' strictly state
diagrams (since the systems may be under kinetic
control) have been published between gelation time
and concentration [S"] and temperature [13]. This
raises the interesting question of the existence (or
otherwise) of the critical gelation concentration and
temperature. Le Bon et al. [S"] criticise Tobitani and
Ross-Murphy for asserting that these must both exist,
although their data [50] do not show them. By con-
trast they claim that 'the temperature dependence is
controlled by an activation energy, and there is no
indication for a critical temperature' other than the
Concentration
Fig. 3. Sol/gel diagram: state ('phase') LCST diagram for a typical
heat-set gelation. The limit values on the concentration and tem-
perature axes correspond to the critical gel concentration C , and
the protein denaturation temperature, T , , respectively.
192 W S . Gosal, S.B. Ross-Murphy /Current Opinion in Colloid & Interface Science 5 (2000)188-194
constraint that gelation will take too long at lower
temperatures [8"]. This is certainly an interesting
observation, but appears to divorce work on heat-in-
duced protein aggregation work from the very exten-
sive work on protein unfolding and co-operativity.
Another controversy concerns the analysis of the
gel modulus by fractal models. Following Bremer and
Shih [56,57], the exponent n , defined above, can be
used to extract a fractal dimension which can, in turn,
be correlated with the df value established by, for
example, scattering studies. Although this approach
has been applied [53',54], we regard it as largely
misconceived. Unfortunately realistic values of df (say
-
1.8-2.4) are only obtained when n 1.5-3, although,
as discussed earlier, firstly no single power law should
be obtained, and secondly, much higher values of IZ
are obtained close to C,. This reflects several factors,
which have recently been enumerated [52], including
that measurements are made too far from the critical
gel concentration (percolation threshold) and that
they are carried out over only a small range of con-
centrations (nearly always less than half a decade).
-
The limit exponent n is anyway reached only when
C > lOC,. Indeed as we have shown recently, [52]
high power laws are obtained for n , even for fibrillar
gels where df must be 5 1.7.
There are several possible explanations for the fail-
ure of the fractal model for the gel modulus [581.
While the dimension d, does appear to be a useful
descriptor for the scattering behaviour of some pre-gel
aggregates, especially far from PI, this may suggest
that the assumptions of, say, the DLCA model regard-
ing reversibility of interactions, are appropriate be-
fore gelation but not afterwards [55'1. Another is
that, since gel structure does not change significantly
post-gel formation while the modulus continues to
increase, the model is simply inappropriate. Finally, as
has been asserted [41,58], at the gel point, only a part
of the protein in the dispersion contributes to the gel
network. This is expected for a percolating system,
but not for a DLCA or RLCA fractal aggregate.
5. Mixed protein gels
Finally we summarise the progress made on studies
of mixed gels. Although there is an increasing amount
of work on globular protein-polysaccharide gels [59],
we have concentrated on mixed gels prepared from
a-lactalbumin (a-La), P-Lg and BSA [60']. Again, the
majority of this work was on crude systems, and was
of little generic applicability. However, a view does
prevail that, e.g. with a-La-P-Lg mixed gels [17,61]
there is some form of synergic interaction, because
p-Lg gels can form at lower concentrations in the
presence of a-La. Recent results found by the au-
thors, however, suggest that all results (gel time and
modulus vs. composition) can be explained simply in
terms of the total protein concentration [19]. In other
words, when partially unfolded, the results obtained
were consistent with a non-specific mixed network
formation between protein species, rather than any
synergic interaction between the individual protein
gel systems. Whether this mixed network formation is
by cross-interaction of fibrils of segregated types, i.e.
P-lactoglobulin and a-lactalbumin, or by the forma-
tion of mixed fibrils from the two unfolded species
remains to be established. It is possible that immuno-
chemical staining techniques could assist here.
References and recommended reading
of special interest
DO of outstanding interest
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Classic reference, although now mostly of historic interest.
[2] Gilsenan PM, Ross-Murphy SB. Viscoelasticity of thermore-
versible gelatin gels from mammalian and piscine collagens. J
Rheol 2000;44:871-883.
[3] Clark AH. Biopolymer gels. Curr Opin Colloid Interface Sci
1996;1:712-717.
Covers papers published on both polysaccharide and protein gels;
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[4] Clark AH. Gelation of globular proteins. In: Hill SE, Led-
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Macromolecules, 2nd ed. Gaithersburg, U S A Aspen Publish-
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Updated version of a chapter that has proved a 'citation classic' in
the area; provides a good coverage of techniques and systems.
Arguably the first call for all new workers in the field.
Ross-Murphy SB. Reversible and irreversible biopolymer gels
- structure and mechanical properties. Berichte der Bun-
sen-Gesellschaft - Phys Chem Chem Phys 1998;102
1534-1539.
Allain AF, Paquin P, Subirade M. Relationships between
conformation of P-lactoglobulin in solution and gel states as
revealed by attenuated total reflection Fourier transform
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de Kruif KG, Hoffmann MAM, van Marle ME et al. Gelation
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Le Bon C, Nicolai T, Durand D. Kinetics of aggregation and
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Valuable and interesting paper, illustrating this French group's
significant contribution to the area, over the last few years. Here,
size exclusion chromatography, light scattering and oscillatory shear
rheology are all employed. Aspects of the discussion at variance
with our views - see text for details.
[9] Hoffmann MAM, Vanmil PJJM. Heat-induced aggregation of
P-lactoglobulin as a function of pH. J Agric Food Chem
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[lo] Baldini G, Beretta S, Chirico G et al. Salt-induced associa-
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Emphasis on monomer-dimer studies, but multi-technique and
good quality data. Data collected both at low pH and at different
ionic strengths. More rigorous use of electrostatics than is usual in
this area.
 WS.Gosal, S.B. Ross-Murphy /Current Opinion in Colloid & Integace Science 5 (2000)188-194
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Very good contribution, sound experimental data and useful his-
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Multitechnique (light, X-ray, neutron) scattering approach applied
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DO drophobic domains in peptides that undergo transformation
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The authors introduced a ‘hydrophobic defect’ into coiled-coil
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Interesting ‘layman’s guide’ to BSE, the prion hypothesis and other
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193
range of biophysical techniques, including rheological. Having stud-
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criterion.
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