[23] P R E C I P I T A T I O N OF P R O T E I N S W I T H P E G 301

[23] P r e c i p i t a t i o n of Proteins with Polyethylene Glycol

B y KENNETH C. INGHAM

This chapter updates a chapter on protein precipitation using polyeth-

ylene glycol that appeared in a previous volume in the series. 1 The use of
nonionic, water-soluble polymers, in particular polyethylene glycol
(PEG), for fractional precipitation of proteins was introduced in 1964 by
Poison et al. 2 Papers appearing over the next two decades provided an
improved understanding of the molecular basis of the protein-precipitat-
ing action of P E G and additional documentation of the unique advantages
of this polymer o v e r other reagents used for this purpose. Although m u c h
of the literature on this subject deals with purification of proteins from
blood plasma, 3 the approach is applicable to any complex mixture. The
principles involved have been clarified by studies with purified proteins,
and the purpose of this chapter is to summarize briefly these principles
with emphasis on practical information enabling the reader to assess the
potential applicability of this technique to specific separation problems.

Advantages of Polyethylene Glycol

The advantages of P E G as a fractional precipitating agent stem primar-
ily from its well-known benign chemical properties. Unlike ethanol and
other organic precipitating agents, P E G has little tendency to denature or
otherwise interact with proteins even when present at high concentrations
and elevated temperatures. Careful experiments designed to test this prin-
ciple revealed that P E G 4004 at concentrations up to 30% (w/v) had no
detectable effect on the circular dichroic spectrum or thermal denatura-
tion temperature of ribonuclease. 5 Subsequent studies confirmed this
result for ribonuclease but suggested that P E G has a destabilizing effect
with some proteins at elevated temperature. 6 This should be of no con-
i K. C. Ingham, this series, Vol. 104, p. 351.
: A. Polson, G. M. Potgieter, J. F. Largier, G. E. F. Mears, and F. J. Joubert, Biochim.
Biophys. Acta 82, 463 (1964).
3 y. L. Hao, K. C. Ingham, and M. Wickerhauser, in "Methods of Protein Fractionation"
(J. M. Curling, ed.), p. 57. Academic Press, New York, 1980.
4 PEG, Poly(ethylene glycol), poly(ethylene oxide), polyoxyethylene. Chemical formula:
HOCH2CHz(CH2CH:O)nCH2CHEOH.PEG 400 and PEG 4000 signify heterogeneous mix-
tures having nominal average molecular weights of 400 and 4000, respectively.
5 D. H. Atha and K. C. Ingham, J. Biol. Chem. 256, 12108 (1981).
6 L. L. Lee and J. C. Lee, Biochemistry 26, 7813 (1987).

Copyright © 1990by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 182 All rights of reproduction in any form reserved.
302 PURIFICATION PROCEDURES: BULK METHODS [23]

cern when using the polymer in protein purification where elevated tem-
peratures are seldom employed. The low heat of solution and the relative
insensitivity of PEG precipitation curves to minor variations in tempera-
ture eliminate the need for controlling temperature during reagent addi-
tion. Another advantage of PEG over ethanol or ammonium sulfate is the
shorter time required for the precipitated proteins to equilibrate and
achieve a physical state suitable for large-scale centrifugation. The advan-
tages of PEG in facilitating the growth of protein crystals is well docu-
mented. 7

Mechanism of Action
Careful measurements with a variety of purified proteins indicate that
their solubilities decrease exponentially with increasing concentration of
PEG according to Eq. (1),
log S = log So - t i c (1)
where S is the solubility in the presence of PEG at concentration C (%,
w/v) and So is the apparent intrinsic solubility obtained by extrapolation
to zero PEG. 5 Plots of log S vs [PEG] exhibit striking linearity over a wide
range of protein concentration, the slope for a given protein being rela-
tively insensitive to pH and ionic strength, but markedly dependent on the
size of the PEG up to about 6000 Da. The slopes also tend to increase with
increasing size of the protein, reinforcing the popular notion of a steric
exclusion mechanism whereby proteins are concentrated in the extrapoly-
met space, eventually exceeding their solubility limit under the given
solution conditions. Although a quantitative explanation of this behavior
is yet to come, it is clear that, in the absence of specific interactions, the
sequence of precipitation of several proteins in a mixture will depend
primarily on the ratios of their initial concentrations relative to their re-
spective solubilities in the absence of PEG. Thus, even though larger
proteins have steeper slopes, a large protein initially present at high con-
centration could precipitate later than a small one present at low concen-
tration if the intrinsic solubility of the latter is much less than that of the
former. Manipulation of the solution conditions is expected to improve
the separation of a given pair of proteins to the extent that their intrinsic
solubilities diverge.

Selection of PEG
Most workers use material with a nominal average molecular weight in
the 4000-6000 range. Polymers larger than this offer no advantage, since
[23] PRECIPITATION OF PROTEINS WITH P E G 303

their solutions are more viscous and the precipitation curves are not much
different from those obtained with PEG 6000. 2,5 Decreasing the molecular
weight below 4000 spreads the precipitation of a mixture over a broader
range of PEG concentrations. The improved resolution that might be thus
anticipated is partially offset by the shallower slopes obtained for individ-
ual proteins. Nevertheless, Honig and Kula 8 found the degree of purifica-
tion of 7-glucosidase from yeast extract to be about 2-fold greater with
PEG 400 than with PEG 4000 or 6000. That PEG 400 is a liquid at room
temperature whose solutions are substantially less viscous than those of
the higher polymers, coupled with the potentially greater ease of remov-
ing it by molecular sieve methods, indicates a need for further compar-
isons.

Analytical Precipitation Curve

The following simple experiment is designed to quickly overcome ig-
norance about the amount of PEG required to precipitate a given pro-
tein(s) from a complex mixture. The scale of this experiment is dictated
by the sensitivity of the assay employed; the availability of a radiolabeled
tracer is a definite advantage. One dispenses a fixed amount (0.1-0.5 ml)
of the mixture into a series of tubes (preferably in duplicate) to each of
which is subsequently added an equal volume of buffer containing in-
creasing amounts of PEG to produce a final concentration of 25-30% in
the most concentrated tubes. It is important to buffer the PEG stock
solutions to avoid PEG-induced changes in pH. 5,9 The increment in PEG
concentration is arbitrary, but 3% (w/v) is adequate for initial screening.
The vigor with which one mixes these solutions depends on the extent to
which the desired protein(s) can withstand mechanical stress; gentle agi-
tation on a vortex mixer is one approach. After 0.5-1.0 hr of incubation at
room temperature or on ice, the samples are centrifuged and the percent-
age of the desired activity remaining in the supernatant liquid is deter-
mined. Inspection of the resulting "analytical precipitation curve" pro-
vides an estimate of the maximum concentration of PEG that can be
added at one time without precipitating the protein of interest as well as
the minimum concentration required to bring it out of solution, parame-
ters that can then be more precisely defined with a second experiment that
focuses on the relevant concentration range. With luck, the curve will fall
either far to the left or far to the right on the PEG axis, defining a simple
7 A. McPherson, Jr., J. Biol. Chem. 251, 6300 (1976).
8 W. Honig and M.-R. Kula, Anal. Biochem. 72, 502 (1976).
9 G. Eichele, D. Karabelnik, R. Halonbrenner, J. N. Jansonius, and P. Christen, J. Biol.
Chem. 253, 5239 (1978).
304 PURIFICATION PROCEDURES: BULK METHODS [23]

one-step method for removing a large portion of unwanted macromole-

cules and/or concentrating the desired activity prior to further processing
by other methods. Otherwise, it may be necessary to obtain a "PEG cut"
via two precipitation steps utilizing in turn the maximum and minimum
concentration of PEG referred to above. It is always possible to manipu-
late the precipitation curve horizontally along the PEG axis by varying
solution conditions.
For screening purposes, it is expedient to choose a fixed concentration
of PEG that causes approximately 50% precipitation of the desired pro-
tein under a given set of solution conditions in order to determine rapidly
the extent to which altering conditions such as pH and ionic strength
might enhance or inhibit precipitation. The most gratifying result of this
approach would be to identify substances or conditions that selectively
influence the solubility of the desired protein. This concept is further
developed in the following section.

Influence of Protein-Protein and Protein-Ligand Interactions

Studies with purified self-associating and heteroassociating proteins
have shed some light on the role of protein-protein interactions on solu-
bility in the presence of P E G ) ,1°-12 Based on the above-mentioned ex-
cluded volume considerations, one predicts that conditions that foster
protein association should enhance precipitation because of the larger
size of the complexes, wheres those that inhibit association would have
the opposite effect. This is the case with almost all systems that have been
examined. Of particular relevance in the present context was the observa-
tion 1° that bovine liver glutamate dehydrogenase at 2.8 mg/ml in 0.2 M
potassium phosphate at pH 7.0, conditions known to promote extensive
self-association, was quantitatively precipitated by PEG 4000 at concen-
trations above 15% (w/v). Such precipitation was completely inhibited,
even at higher concentration of PEG, by the combined presence of l0 -3 M
N A D H and GTP, cofactors known to reverse the self-association. Similar
effects were observed with chymotrypsin, chymotrypsinogen, and fl-lac-
toglobulin A, in which cases self-association was manipulated by varying
pH and ionic strength, parameters likely to be less selective. Nonspecific
electrostatic interactions between oppositely charged proteins such as
albumin and lysozyme can also have profound effects on solubility that
are most pronounced at low ionic strength at a pH between the pI of each
of the two proteins.ll While such interactions are frequently viewed as a

i0 S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 191, 525 (1978).

11 S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 203, 630 (1980).
12 j. Wilf and A. P. Minton, Biochim. Biophys. Acta 671}, 316 (1981).
[23] PRECIPITATION OF PROTEINS WITH P E G 305

nuisance, to be minimized by maintaining near-physiological ionic

strength, the possibility of using them to advantage in a purification
scheme should be kept in mind.
A more specific type of heteroassociation of the type that might be
exploited in purification is the functional interaction between human
plasma fibronectin and denatured collagen, i.e., gelatin. The precipitation
curve for the plasma protein in phosphate-buffered saline shifted from
11% PEG to less than 3% PEG upon addition of gelatin, which by itself
was not precipitated by PEG under these conditions. 13Since the complex
between the two proteins is very stable, even at high ionic strength, it
should be possible to precipitate fibronectin selectively from a complex
mixture by this method. The contaminating gelatin could then be re-
moved, e.g., by ion-exchange chromatography in the presence of urea.
Although the advantage of this approach over affinity chromatography on
immobilized gelatin is debatable, the example serves as an additional
illustration of the application of bioaffinity principles to fractional precip-
itation. Any substance that interacts specifically with the desired protein
has the potential to alter its solubility selectively and should thus be
tested. Enzymes are ideal candidates for this approach, since they often
interact with one or more effectors or cofactors, sometimes with large
changes in the state of association.

Methods of Removing PEG

In many applications, PEG is used early in the purification scheme and
is removed during subsequent chromatographic steps on ion-exchange or
affinity columns to which PEG has no tendency to absorb. A word of
caution is in order regarding the application of PEG-containing solutions
to some exclusion columns, the performance of which can be significantly
altered owing to osmotic effects of the polymer. 14Alternative approaches
to removing PEG include ultrafiltration ~5,16and salt-induced phase separa-
tion ~7as reviewed) 8 The latter method is particularly useful for solutions
containing relatively high concentrations of PEG and has the potential
advantage that the protein may be concentrated in a low-volume, salt-rich
phase. For many research purposes it is probably unnecessary to remove

t3 K. C. Ingham, S. A. Brew, and S. I. Miekka, Mol. lmmunol. 20, 287 (1983).

14 K. Hellsing. J. Chromatogr. 36, 170 (1968).
15 T. F. Busby and K. C. Ingham, J. Biochem. Biophys. Methods 2, 191 (1980).
16 K. C. Ingham, T. F. Busby, Y. Sahlestrom, and F. Castino, in "Ultrafiltration Membranes
and Applications" (A. R. Cooper, ed.), p. 141. Plenum, New York, 1980.
17 T. F. Busby and K. C. Ingham, Vox Sang. 39, 93 (1980).
~s K. C. Ingham and T. F. Busby, Chem. Eng. Commun. 7, 315 (1980).
306 PURIFICATION PROCEDURES: BULK METHODS [23]

all traces of polymer from the final product, since it is optically transpar-
ent 19 and helps prevent loss of protein by absorption of glass.

Summary
Polyethylene glycol is a nondenaturing water-soluble polymer whose
ability to precipitate protein from aqueous solution can be qualitatively
understood in terms of an excluded volume mechanism. The increment in
PEG concentration required to effect a given reduction in solubility is
unique for a given protein-polymer pair, being insensitive to solution
conditions and primarily dependent on the size of the protein and poly-
mer. Selective manipulation of the solubility of specific proteins through
control of their state of association or ligand environment can potentially
remove some of the empiricism otherwise involved in fractional precipita-
tion. Adequate methods for removing the polymer are available.

19 T h e low level o f U V a b s o r b a n c e frequently found in s o m e PEG preparations is not

inherent to the p o l y m e r but is due to a small a m o u n t o f antioxidant s o m e t i m e s added by
the m a n u f a c t u r e r .