ELSEVI E R Current Opinion in Colloid & Interface Science 5 (2000) 38-43

www.elsevier.nl/locate/cocis

Interactions and phase transitions in protein solutions

Roberto Piazza*
Unit; INFM, Dipartimento di Ingegneria Nucleare, Politecnico di Milano, 20133 Milano, Italy

Abstract

Understanding interactions and phase transitions in protein solutions is essential in order to develop systematic protein
crystallisation strategies. Recent studies show that proteins near crystallisation behave as particles interacting via a very
short-range attractive potential. The microscopic origin of this attractive term, and its strong dependence on the nature of the
crystallisation agent, is, however, still obscure. The interplay of crystal nucleation with formation of weakly bonded amorphous
aggregates also deserves further analysis. 0 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Proteins; Colloidal suspensions; Complex fluids; Phase transitions; Crystallization; Light scattering; Aggregation

1. Introduction phiphilic molecules self-aggregation. We will mainly

concentrate on a few significant analogies:
Obtaining good-quality crystals is a fundamental
step in order to study 3-D protein structure by means
. Protein crystallisation is generally performed at
quite high ionic strength, where the interparticle
of X-ray diffraction, but is far from being easy.' interaction potential is very short-ranged and
Therefore, understanding the nature of protein inter-
strongly related to hydration properties connected
actions in solution and predicting their phase be-
to the hydrophilic-hydrophobic 'patching' of the
haviour is of extreme practical interest. Globular pro-
teins are colloidal macroions, with a variable charge
trimmed by the solution pH, and therefore share
general features in common with simple charged latex
. protein surface.
The phase diagram of a protein solution, as much
as many properties of amphiphilic solutions, is
particles. However, since they are hydrophilic colloids extremely salt-specific even for monovalent salts.
and form thermodynamically stable solutions in water, The relative efficiency of different ions scales as
some of the crucial traits of protein solution be- their effect on micellar cmc, structure and cloud
haviour are more related to similar aspects of am-
. points.
Amorphous aggregates seem to form in supersatu-
rated solution and hinder, but possibly also pro-
mote, crystal growth. At variance with salt-induced
+ +
* Tel.: 39-2-2399-6386;fax: 39-2-2399-6309. colloidal aggregation, such aggregation pheno-
E-mail address: roberto.piazza@polimi.it (R.Piazza).
'For a general review on physical chemistry of protein solutions mena are reversible, and aggregates are prone to
and protein crystallisation see, for instance Ducruix and Gieg [l]. relevant restructuring.

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 ) O O O 3 4 - 0
 R Piazza /Current Opinion in Colloid & Interface Science 5 (2000)38-43
We mainly review the work done in the last 2 years,
although reference to earlier fundamental contribu-
tions to understanding protein interactions and phase
diagrams is made whenever needed.
2. From phase diagrams to the nature of the
interaction potential
Protein solutions present very peculiar phase dia-
grams. At fixed pH, temperature and concentration of
a 'precipitant', or crystallising agent (very often a
simple salt) are the relevant controlling parameters.
Back in the late 1970s, pioneering experiments by
G. Benedek group in MIT [2] proved that the phase
diagram of some protein shows a liquid-liquid coexis-
tence curve. However, later studies by Broide et al. [31
and by Mushol and Rosemberger [4] pointed out that
liquid-liquid phase separation is actually metastable
with respect to crystallisation, and that crystal nucle-
ation rapidly takes place within the droplets of the
concentrated phase. This situation is intriguing. A
colloidal suspension can be mapped onto an equiva-
lent one-component fluid of particles interacting via
an effective potential: liquid-liquid phase separation
is then the equivalent of a gas-liquid transition, while
the solubility curve parallels to the freezing line.
Therefore, a protein solution behaves as a simple
substance presenting one singZe stable fluid phase due
to 'sinking' of the critical point below the freezing line
[5']. Hagen and Frenkel [6] have shown that this
situation is typical for interaction potentials consist-
ing, in addition to an excluded volume contribution,
of an attractive term with a range much shorter than
the hard particle diameter. Similar interactions, which
do not pertain to simple fluids, are typical for instance
of depletion forces in colloidal suspensions. Rein ten
Wolde and Frenkel [7"] have pointed out that the
proximity of a critical point, although metastable, is
expected to notably speed up the nucleation kinetics,
a fact actually observed in lysozyme crystallisation
[4,8]. An additional feature of short-range interactions
is that freezing is often hindered by the formation of
gels, as very often observed in colloid/polymer mix-
tures. This is unluckily also the end point of many
attempts to crystallise proteins. The question is there-
fore why protein phase diagrams share such features.
The classical DLVO interaction picture applies to
protein solutions at low ionic strength [9], but it is
intrinsically unable to capture the main features of
protein behaviour in salting-out conditions, that is
when large amounts of electrolyte are added to in-
duce crystallisation [lo]. A vast amount of experimen-
tal evidence presently suggests that the leading con-
tribution to protein interactions near crystallisation is
a very short-range attraction. Rosenbaum et al. [ll"]
39
have used the theoretical expression for the second
virial coefficient, B,, of adhesive hard-spheres A H S
(hard-spheres interacting via an attractive potential of
vanishing range to successfully map their light-scatter-
ing data on the phase diagram predicted in Hagen
and Frenkel [6] for a very short-range Yukawa poten-
tial. This also explains why B, values near crystallisa-
tion seem to fall in a very narrow range [12]. Piazza
et al. [13'] measured by static light scattering the
osmotic compressibility of lysozyme up to very high
protein concentration showing an excellent agree-
ment with the theoretical expression for the osmotic
equation of state of A H S . Although the AHS model
has been often criticised because of its thermody-
namic inconsistency, it should be noticed that this
rigorous result holds only for extremely monodisperse
spherical particles [14]. Specific features of the phase
diagram like the exact location of the consolution
curve, which is very sensitive to the detailed form of
the interactions [15], require for sure more refined
potentials, which could be extracted from the full
structure factor, S(q). This is not easy when dealing
with short-range potentials, since the first peak of
S ( q ) is partly masked by the concomitant strong decay
of the form factor. Recent X-ray measurements [16']
confirm anyway the need for a very short-range at-
tractive term. Nor0 et al. [17'] have recently sug-
gested that an additional Van der Waals long-range
term should be introduced to account for the effective
occurrence of liquid-liquid phase separation, which is
conversely always hindered by the onset of gelation in
depletion-induced colloid phase separation. Isotropic
potentials are in any case intrinsically unable to de-
scribe crystal nucleation and growth, and anisotropic
interactions (physically related to hydrophilic-hydro-
phobic 'patches' on the particle surface) might also
change the overall nature of the phase diagram
[18',19]. One of the most peculiar aspect of protein
interactions is the strong correlation between osmotic
virial coefficient and solubility, S . Guo et al. [20']
have shown that an impressive set of experimental
data for different proteins in different conditions fall
on a single ( S , B 2 ) master curve. A possible explana-
tion for this experimental correlation has been given
by Haas et al. [21] on the basis of a simple form for
the free energy of a protein solution [22].
3. Salt-specificity and the Hofmeister series
Although we do have indications on the range and
strength of attractive interaction potential induced by
'salting-out' proteins with electrolytes, its origins are
still mysterious. Indeed, the salting-out effect strongly
depends on the kind of electrolyte. In particular,
protein solubility varies strongly with the specific an-
40 R. Piazza /Current Opinion in Colloid & Inteflace Science 5 (2000)38-43
ion composing the added electrolyte, and the so-called
Hofmeister series sets a well-defined empirical scale
of efficiency for different ions in precipitating pro-
teins. Besides its effects on protein solubility, the
Hofmeister series sets the trend of an impressive
number of physical properties of electrolyte solutions
[23]. It is well known that ion-specificity plays also a
mayor role in determining aggregate structure and
interactions in amphiphilic solutions, the simplest ex-
amples being for instance the striking differences
between CTACl- and CTABr- micelles, or counte-
rion effects on swelling of lamellar phases. The
Hofmeister series controls moreover the location of
the consolution curve of non-ionic surfactants2 and
cmc, clouding, and gelation of block-copolymer aggre-
gates.3 However, in spite of its evident relevance in
the study of aqueous solutions, the Hofmeister series
has not been given so far a clear microscopic explana-
tion. Detailed studies on lysozyme [26] have shown
that inorganic and organic anions strongly affects
solubility following the Hofmeister series in a reverse
order compared to most proteins. Since lysozyme has
in most experimental conditions a positive charge
(most proteins are negative at normal pH), ordering is
probably related to the sign of surface charge [27].
Solubility is, however, a macroscopic quantity, and the
question is how the Hofmeister effect is related to
interparticle interactions. Ducruix et al. [28] have
shown that the structure factor of lysozyme solutions
near crystallisation is strongly affected by the nature
of the precipitant electrolyte. Piazza and Pierno [29]
performed a study of lysozyme osmotic virial
coefficients as a function of salt concentration for a
well-defined series of monovalent salts, yielding quan-
titative coefficients for the effectiveness of different
anions and cations. Lysozyme (a very easily crystallis-
able and rather peculiar protein) might not be the
whole story. Recent results [30] suggest that in horse
apoferritin solutions, the addition of large amounts of
electrolytes give rise to repulsive interactions. Similar
effects are not new, and are needed to explain the
exceptional stability to coagulation of protein-coated
colloidal particles at high ionic strength [31]. The
origin of the Hofmeister series is surely related to the
effects of electrolytes on aqueous interfaces. Elec-
trostatic effects are known to strongly alter water/air
interfacial tension4 and a close relation between salt-
ing-out coefficients and surface tension increment has
been noticed long ago by Melander and Horwath [33].
Ninham and Yaminsky [34] have, however, shown
that, at high ionic strength, dispersion (elec-
*See, for instance: Schott [24] and references therein.
3See, for instance: Pandit et al. [25] and references therein.
4See for instance: Palauch [32] and references therein.
trodynamic) interactions between counterions and
charge colloids are larger than electrostatic terms,
and cannot be treated separately as in the DLVO
theory. Although Lifshitz dispersion theory does pre-
dict a very different role for cations and anions, due
to their different polarizability, a (perhaps very simpli-
fied) form for a fully electrodynamic interaction po-
tential is still lacking. Direct studies of the interaction
potential by scanning probe or surface force measure-
ments are probably imperative for better quantitative
understanding [35].
4. Polymer-induced crystallisation
Besides electrolytes, polymers, and in particular
polfiethylene glycol) (PEG), are commonly used as
precipitants in protein crystallisation. It seems
straightforward to associate polymer-induced protein
crystallisation to depletion effects similar to those
seen in polymer-colloid mixtures. This has been in-
deed done a decade ago by Mahadevan and Hall [36]
on the basis of a simple equivalent one-component
model. However, the protein case is very different
from colloidal depletion flocculation. PEG and pro-
tein molecular weights are often comparable, and
polymer concentration is generally well beyond the
semidilute regime. Recently Chatterjee and Schweizer
[37,38] have developed a theory of polymer depletion
effects on dilute suspensions of spheres, valid for all
size ratios, deriving a potential of the mean force
which depends on the mesh size of a semidilute
polymer solution. This model seems to partially ac-
count for recent data by Kulkarni et al. [39] on virial
coefficients of lysozyme in PEG solutions. However,
the previous model underestimates strong effects on
the depletion due to the proximity of PEG critical
consolution point, similar to those reported for sur-
factant-induced depletion near the critical point of
non-ionic micellar solution [40]. An open question is
whether also salting-out by electrolytes might be
treated in terms of depletion concepts. Dispersion
forces may indeed be related to ion depletion [341,
and ion-exclusion effects could induce effective at-
tractive forces between neutral surfaces [41]. How-
ever, so far these observations are difficult to quantify
in terms of measurable ion properties, and simply
mean that entropic effects at high salt concentration
might be relevant.
5. Crystal growth precursor aggregates or not?
One of the most controversial experimental topics
is whether the occurrence of amorphous aggregates is
germane or not to crystal nucleation. Georgalis and
 R Piazza /Current Opinion in Colloid & Interface Science 5 (2000)38-43
co-workers [42,43] have reported a huge amount of
experimental evidences of amorphous clusters with a
fractal structure in supersaturated (but not necessar-
ily crystal-forming)lysozyme solutions. However, these
findings have neither been corroborated by light scat-
tering measurements in supersaturated lysozyme solu-
tions [9,13], nor by many optical studies of crystal
nucleation [44]. Tanaka et al. [45] did detect aggre-
gates in lysozyme solution by dynamic light scattering.
However, their size and number density are far too
small to account for the scattering signals reported in
Umbach et al. [421. More experiments are therefore
needed to settle this issue. We simply point out that
requirements of protein purity are more stringent for
aggregation-crystallisation measurements than for so-
lution studies. Batches produced by Seikagaku, Japan,
used for instance in Tanaka et al. [45], are known to
be much purer [461 and yield much more reproducible
solubility data, than those utilised in Umbach et al.
[42] and Georgalis et al. [43]. Moreover, aggregates
forming in supersaturated protein solution are re-
versible (they easily dissolve, for instance, by increas-
ing temperature), and undergo manifest restructuring
(crystals are known to grow from amorphous aggre-
gates over long time scales). Kinetics and aggregate
morphology for reversible colloidal aggregation
change a lot. For instance, it does not matter any
more whether aggregation is diffusion or reaction
limited, and the fractal dimension of the aggregates
becomes time dependent [47].
Aggregates or not, compared to simple molecular
systems proteins solutions sustain much larger super-
saturation before nucleation takes place, and show
very long crystallisation induction times (ranging up
to days or weeks). The origin of this difference may
be due to the high interfacial tension between coexist-
ing phases for deep short-range potential [48,49].
Accurate nucleation rate data (also in proximity of
the metastable critical point), which could be used as
a test for nucleation theories, are now available thanks
to careful measurements by Galkin and Vekilov [501.
6. Conclusions
A big effort to approach protein phase behaviour
using the tools of colloidal science, and to frame it
within basic concepts in condensed matter physics,
has therefore been made in the last few years. Much
is still to be done. Understanding the basic physics of
hydration forces, digging out the roots of salt-specific
interactions, and modelling spontaneous aggregation
and crystal nucleation in solutions of particles inter-
acting via short-range potentials are likely to be among
the mainstreams of future research in this field.
41
Acknowledgements
I thank very much the Advanced Research Project
PROCRY of the Italian Institute for Condensed
Matter Physics (INFM), which led me to get inter-
ested in the problems discussed in this review.
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43
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solid phase of spherical particles with short-ranged attraction.
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In this paper, classical nucleation theory is applied to calculate the
free energy barrier to nucleation for short-range attractive poten-
tials. If the range of the interaction is 1/10 of the particle diame-
ter, the interfacial tension of the nuclei is found to be an order of
magnitude larger than for hard spheres. Therefore, the nucleation
rate at fixed chemical potential difference is strongly reduced.
[49] Sear RP. Metastability and nucleation in the dilute fluid
phase of a simple model of globular proteins. Preprint cond-
mat/9912199.
[50] Galkin 0, Vekilov PG. Are nucleation kinetics of protein
crystals similar to those of liquid droplets? J Am Chem SOC
2000;122:156- 163.
The novel method developed in Galkin and Vekilov [44] is applied
to measure nucleation rates in a wide range of lysozyme crystallisa-
tion conditions, also close to the metastable critical point. Results
are consistent with a very small size for the critical nuclei, and large
statistical fluctuations in the nucleation rate near the critical point
are detected.