ARCHIVES

OF BIOCHEMISTRY

Vol. 186, No. 1, February,

Precipitation

AND

BIOPHYSICS

pp. 106-113, 1978

of Proteins

with Polyethylene

Glycol:

Characterization

of Albumin
KENNETH C. INGHAM
American

National

Red Cross,

Blood Research

Laboratory,

Bethesda,

Received March 15, 1977; revised September

8, 1977

Maryland

20014

The factors which govern the precipitation of albumin by the synthetic polymer,
polyethylene glycol (PEG), have been investigated as part of a broader program to
determine the molecular basis of the protein-precipitating
action of synthetic polymers.
The concentration of PEG4000 required for precipitation of albumin was minimum
near the isoelectric point and was only slightly dependent on temperature between 4
and 30C. At pH > fl (pH 4.5), increasing the ionic strength shified the precipitation
curve to a higher concentration of PEG, with different salts having quantitatively
similar effects. At pH 4, the opposite effect was observed and different salts varied in
their effectiveness according to their position in the lyotropic series. Dilution of
albumin shifted the midpoint of the precipitation curve at pH 4.5 to a higher concentration of PEG with no significant effect on the slope. Variation of the molecular weight of
PEG from 6000 to 400 shifted the precipitation to a higher PEG concentration (percentage, w/v) and diminished the slope of the curves. Precipitation of albumin dimers
required less PEG than did monomers and the separation between the precipitation
curves for monomer and dimer was greater with PEG-400 than with PEG-4000. Labeling
of albumin with 2.3 mol of dansyl chloride had no effect on the precipitation curve at
pH 4.5. The polarization of dansyl fluorescence was measured as a function of PEG
concentration to determine whether albumin self-associates prior to precipitation. The
results indicate that albumin remains in a monomeric state even at a PEG concentration
beyond that required to initiate precipitation, reflecting the highly cooperative nature
of the precipitation process.

The use of synthetic polymers for fractional precipitation of proteins was stimulated by the work of Polson et al. (11, who
pointed out several advantages of polyethylene glycol (PEG) over other water-soluble polymers such as dextran, polyvinyl
alcohol, and polyvinyl pyrrolidone. These
authors went on to develop a scheme for
the purification of y-globulin and fibrinogen from human plasma. Since that time,
an increasing number of reports have appeared describing the purification of additional proteins from plasma (2-12) and
other sources (13-17). PEG has also been
1Abbreviations used: PEG, polyethylene glycol,
polyethylene oxide, polyoxyethylene
[chemical formula, HOCH,CH,(CH,CH20),CH,CH,0Hl;
PEG4000 and PEG-400, heterogeneous mixtures having
nominal average molecular weights of 4000 and 400,
respectively;
dansyl,
dimethylaminonaphthalene
sulfonyl; P, polarization.
0003-9861/78/1861-0106$0!2.00/0
Copyright 0 1978 by Academic Press, Inc.
All rights of reproduction in any form reserved

used to separate free antigens from those

bound to antibody (18) and free hormones
from those bound to membranes (19) or
solubilized receptors (201, thereby facilitating the development of immuno- and
receptor assays.
The selectivity of precipitation was reported to be optimum near room temperature (1) and subsequent studies suggested
that rigorous temperature control was not
important (17, 21). In addition, PEG is
relatively benign with respect to protein
denaturation, especially in comparison
with ethanol, and may even have a stabilizing influence on macromolecular structure (15, 22). Such observations account
for efforts to utilize this polymer in the
large-scale fractionation of plasma proteins (7-12, 23, 24) as an alternative to
cold ethanol (25).
In spite of the increasing interest in

PROTEIN

PRECIPITATION

WlTH

PEG as a fractional precipitating agent,

the factors which govern the precipitation
process are poorly understood. There is a
need for systematic investigation of the
influence of such variables as temperature, ionic composition, and the molecular
weight of PEG. Many of the existing
plasma fractionation schemes have been
derived emperically by stepwise addition
of the precipitating agent and subsequent
analysis of the resulting fractions. While
this approach is adequate for obtaining
limited numbers of products, the development of a more comprehensive scheme
will require a thorough characterization
of each protein in the mixture. With increasing numbers of plasma proteins becoming available in a high degree of purity, such an approach is now feasible.
The objectives of this approach are to determine means for selectively manipulating the precipitation of each protein in the
mixture, thereby facilitating the development of a comprehensive separation
scheme. This report presents the results
of an initial study with albumin, the most
abundant and clinically most important
protein in plasma, as part of a broader
program designed to elucidate the molecular basis of the protein-precipitating action of synthetic polymers.
MATERIALS

AND

METHODS

Fractions of PEG having nominal average molecular weights between 400 and 6000 were purchased
from Fisher Scientific under the trade name Carbowax. They were used without further purification.
A batch of PEG-4000 which was purified by etherinduced precipitation
from benzene was found to
give a precipitation
curve for albumin which was
identical to that obtained with the unpurified
material. Unless otherwise indicated, the data reported
here were obtained using PEG-4000 without further
purification.
Gel filtration
of human albumin (Calbiochem, tryst. B grade) on Sephadex G-100 (1.5 x
75 cm) gave a single peak of absorbance at 280 nm
with a leading shoulder reflecting
the presence of
5-108 dimer, depending on the batch. There was
an additional peak of absorbance in the salt volume
which had a fluorescence spectrum characteristic
of
N-acetyltryptophan,
which is frequently
added to
albumin as a stabilizer.
Experiments
using albumin, from which tryptophan
was removed by dialysis, indicated that the presence of this amino
acid up to a concentration
of 2 x 10ms M had no
effect on precipitation
with PEG at pH 4.5. Unless

POLYETHYLENE

107

GLYCOL

otherwise
indicated,
data reported here were obtained with human albumin which was not further
purified. A few experiments
utilized albumin from
which the dimers were removed by gel filtration.
Bovine albumin
was also from Calbiochem
(A
grade).
Albumin
was labeled with the dansyl chromophore by treatment
with 1 mg of dansyl chloride
(Sigma) per 100 mg of albumin at room temperature,
pH 8.5, for 1 h. The product was passed through a
Sephadex G-100 column to remove unreacted dye.
The elution profile obtained by fluorescence at 500
nm was similar to that obtained with unlabeled
albumin using absorbance at 280 nm, suggesting
that the labeling procedure did not alter the monomer-dimer
composition and that the dansyl chloride did not react preferentially
with either species.
By comparing the absorption spectrum of the product with that of dansyl lysine (Sigma) and using
E 280= 5.4/mg/ml (261, a labeling ratio of 2.3 mol of
dansyl per mole of albumin was estimated.
The
monomer and dimer fractions were further purified
by rechromatography
on Sephadex G-100.
Precipitation
curves were obtained by mixing
0.15-0.3 ml of a stock solution containing
protein,
buffer, and salt, all at twice the desired concentration, with an equal volume of aqueous PEG, also at
twice the desired concentration.
The mixture was
mixed on a Vortex mixer, incubated at room temperature (24 + 1C) for 30-45 min, and centrifuged for
10 min at approximately
3300g. Longer incubation
periods did not cause additional
precipitation.
The
supernate was then analyzed for protein, usually
by measuring the optical density at 280 nm after
appropriate dilution with water. All measurements
were done in duplicate and the averages were converted to percentage
protein in the supernate.
The baseline, corresponding
to complete precipitation, was determined with control samples to which
trichloroacetic
acid was added to a concentration
of
10%. With the undialyzed
human albumin,
the
baseline optical density was about 30% of the total,
due to the presence of N-acetyltryptophan,
which
was not precipitated
by PEG. In spite of this rather
large background,
precipitation
curves were extremely reproducible
and did not differ from those
obtained with albumin from which N-acetyltryptophan was completely removed by dialysis.
Absorption measurements were made on a Cary
118-C spectrophotometer
and fluorescence measurements on a Perkin-Elmer
Model MPF-4 equipped
with a thermostatted
cell compartment
and polarizing filters. Polarization
of fluorescence
was measured at 90 from the vertically
polarized excitation
beam and P was calculated from the relation
P = (I, - GZ,)/U,

+ GZ,),

where Iv and I, are the observed intensities

with
the emission polarizer in the vertical and horizontal

108

KENNETH

C. INGHAM

positions, respectively, and G = 2,/Z, was obtained

using horizontal excitation (27).
RESULTS

Influence of PH. The influence of pH on

the position of the precipitation curve for
albumin is illustrated in Fig. 1. At neutral
pH, concentrations of PEG greater than
25% were required for appreciable precipitation. Lowering the pH shifted the precipitation curve steadily to a lower PEG concentration until a pH of approximately
4.5. Further lowering of the pH caused
the curve to shift back to the right. The
dependence of the midpoint of the precipitation curve on pH is illustrated by the
squares in Fig. 2.
At a given concentration of PEG, the
solubility of albumin was extremely sensitive to pH. This is also illustrated in
Fig. 2 for human and bovine albumin in
11% PEG, where both proteins exhibit a
sharp minimum near their isoelectric
point (26). Under these conditions, a
change of only 0.1 pH unit can drastically
alter the solubility.
Influence of temperature.
A potential
advantage of PEG precipitation as a largescale method of fractionating plasma proteins is the possibility of working at room
temperature without the need for rigorous
temperature control (21). Precipitation
curves were obtained under the conditions
of Fig. 1 at pH 4.5 and 7.1 at 4C, room
temperature, and 30C. The midpoints of

10

15
20
% PEG

25

30

FIG. 1. Influence of pH on the precipitation of

albumin by PEG4000 at room temperature. The
concentration of albumin was 20 mg/ml. The buffer
was 0.05 M potassium phosphate at pH 7.1 and 0.05
M potassium acetate at a lower pH. All solutions
contained 0.1 M. KCI.

I .

J
7

:0

5
w
a
II

PH

FIG. 2. Influence of pH on the solubility of human (0) and bovine (A) albumin at room temperature in the presence of 11% PEG-4000, 0.05 M potassium acetate, and 0.1 M KCl. In all cases, the initial
protein concentration
was 20 mg/ml. The lower
curve (Cl) illustrates the pH dependence of the
midpoint of the precipitation curve.

the precipitation curves at pH 4.5 were

7.3, 8.7, and 9.2% PEG, respectively. At
pH 7, the midpoints varied between 27
and 30% PEG. There was no significant
difference in the shape of the curves. It
was thus concluded that varying the temperature is not a useful means of manipulating the precipitation of albumin and
that minor fluctuations around room temperature would not have a significant effect on the precipitation. This latter point
was confirmed by results obtained at room
temperature over a period of 1 year, during which the midpoint of the precipitation
curves at pH 4.5 did not vary by more
than 0.3% PEG. Juckes (21) found that
the precipitation of bovine albumin by
PEG-6000 shifted only about 1% between
4 and 22C.
Influence of ionic composition.
At pH
values equal to or above the isoelectric
point (pH approximately 4.5), the addition
of salts inhibited the precipitation of albumin by PEG-4000. For example, in 0.05 M
K-acetate buffer, pH 4.5, the addition of
0.5 M KCl, KSCN, or KPO, shified the
midpoint of the precipitation curve from
8% to approximately 17% PEG. There was
very little difference between the effects
of the three salts. By contrast, at pH 4,
below the isoelectric point, the addition of
salts enhanced the precipitation, and different salts varied considerably in their
effectiveness. To screen a variety of salts,

PROTEIN

PRECIPITATION

WITH

their effects were determined under a single set of conditions, namely, 0.05 M Kacetate buffer, 11% PEG, and pH 4.0. The
results are illustrated in Fig. 3. With few
exceptions, their effects correlate with
their position in the Hofmeister, or lyotropic, series (28). One of the exceptions
was the sulfate anion. Under these conditions, the addition of potassium or ammonium sulfate to a concentration of only
0.02 M caused approximately 90% precipitation of albumin. Although ammonium
sulfate has long been used as a precipitating agent, its effects are generally express& at a much higher concentration.
The origin of its unusual potency under
these particular conditions could be related to the N-F transition which albumin
undergoes in acid with the concomitant
exposure of hydrophobic residues normally
buried on the interior of the protein (29,
30).
Influence

of albumin concentration.
As
the initial concentration of albumin is de\

POLYETHYLENE

GLYCOL

109

creased, the concentration of PEG required for 50% precipitation is increased,

as illustrated in Fig. 4. However, there
was no significant change in the shape
(i.e., the steepness) of the precipitation
curves, even over a 50-fold range of initial
albumin concentration. Thus, dilution of
the solution does not appear to offer a
practical means of sharpening the precipitation curves for albumin. It should be
pointed out that the data corresponding to
the filled circles in Fig. 4 were obtained
by the addition of solid PEG to saturated
solutions, all of which were in contact
with excess albumin. Thus, even under
conditions of saturation, PEG up to a concentration of 4% had no effect on the
solubility, i.e., it was neither diminished
nor enhanced. Furthermore, there was no
visual indication of the formation of two
liquid phases, as reported by Edmond and
Ogston (31).
The data in Fig. 4 in the range of 1314% PEG provide multiple determinations
of the absolute solubility of albumin under
those conditions. In the experiments corresponding to initial concentrations of 20,
5, and 2 mg/ml, the respective concentrations of albumin in the 13% supernates
were1.5, 1.6, and 1.5 mg/ml. The corresponding values in the 14% supernates
were 0.70, 0.82, and 0.83 mg/ml. This good
agreement indicates that the concentra100
Q)
;;i
E 80
:

z
.E

01

60

02
03
0.4
05
Salt Concentration
IMI

FIG. 3. Influence of neutral salts on the solubility of albumin in the presence of 11% PEG and 0.05
&Mpotassium acetate at pH 4.0 and room temperature. In A, the cation was potassium except in the
c:l ;e cl sulfate, where both potassium (A) and ammonium (0) were used. In B, the anion was chloride. The initial concentration
of albumin was 20
mgiml.

% PEG
Fro. 4. Influence of albumin concentration
on ita
precipitation
by PEG-4000. The solutions contained
0.05 M potassium acetate, 0.1 M KC1 at pH 4.5 and
room temperature.
The concentrations
of albumin
were: 0, saturated (approximately
125 mg/ml); 0,
20 mg/ml; 0, 5 mg/ml; A, 2 mg/ml. The latter three
curves were obtained using purified monomers.

110

KENNETH

tion of albumin remaining in the supernates was not dependent on the amount of
precipitate formed and suggests that thermodynamic equilibrium
was achieved. In
support of this conclusion was the observation that experiments
using different
modes of mixing and extended incubation
periods gave identical results.
Effect of molecular weight of PEG. The
concentration of PEG required for precipitation of albumin increases as the degree
of polymerization
decreases (Fig. 5). There
is also a tendency for the curves to
broaden, especially in the initial and final
stages of precipitation.
Thus, a 4% increment in the concentration of PEG-6000 is
required to go from 10 to 90% precipitation,
while, with PEG-400, a 10% increment is
required. These observations are similar
to those reported for y-globulin by Polson
et al. (l), who also included PEG-20,000 in
their study and found it to be about as
effective as PEG-6000.2 However, solutions
of the higher molecular weight polymer
are discolored and viscous and thus less
convenient to work with.
Comparison of monomers and dimers.
The size of a protein is believed to be an
important factor in governing its precipitation by synthetic polymers (21). This
conception is based primarily on gross correlation of data obtained on a variety of
proteins differing not only in size but in
other properties as well. To test this hypothesis under conditions where variables
other than size are reasonably constant,
albumin dimers were isolated from commercial albumin by chromatography
on
Sephadex G-100 (see Materials and Methods). Precipitation
curves were then obtained under identical conditions for the
monomer and dimer, using both PEG-4000
and PEG-400. The results are given in
Fig. 6. With both PEGS, the precipitation
2 We found PEG-2OM to be slightly less effective
than PEG-4000, giving 50% precipitation
at a concentration of 9.5%. However, PEG-2OM as manufactured by Union Carbide is not simply a higher
member of the homologous series, HO(CH,CH,O),H,
but a mixture, prepared by coupling 2 mol of PEG6000 with an epoxide (39). This fact is not always
clearly stated and our analyses indicate that material marketed
by several distributors
under the
label PEG-20,000 is in fact PEG-2OM (22).

C. INGHAM

*PEG

FIG. 5. Influence of the molecular weight of PEG

on its precipitation
of albumin in 0.05 M potassium
acetate, 0.1 M KCl, pH 4.5. The initial concentration
of albumin was 20 mg/ml.

m IOO;i
E
g so2
E
-E
2
Q
E
8

60.
m20.

d
O-

10

I5
Percent

20
PEG

25

30

FIG. 6. Comparison of the precipitation

of albumin monomers (0) and dimers (0) by PEG-4000
(dashed curves) and PEG-400 (solid curves) under
conditions identical to those of Fig. 5.

curves for the dimer are shifted to the left

and the dimer is BO-90% precipitated at a
concentration of PEG where precipitation
of the monomer is first detected. The separation between the monomer and dimer
curves is greater with PEG-400 than with
PEG-4000. Precipitation curves for the unfractionated albumin (see Fig. 5) are essentially what would be expected for an
appropriate mixture of monomer and dimer, assuming the effects to be additive.
Note that the onset of precipitation
is
sharper with the purified material.
Dansyl-labeled
albumin. As part of our
investigation
of the molecular mechanism
of precipitation
by nonionic polymers, it
was of interest to explore the influence of
added polymer on the state of self-association of the protein. Although albumin is
not considered to be a reversibly self-associating protein (32), it is conceivable that
some form of self-association
might precede actual precipitation.
Does albumin

PROTEIN

PRECIPITATION

WITH

persist in a monomeric state upon addition

of PEG, or does it gradually form dimers,
trimers, and higher oligomers until the
point of precipitation is reached? To answer this question, albumin was labeled
with the fluorescent probe, dansyl chloride, and the labeled monomers and dimers were purified by gel filtration (see
Materials and Methods). The objective was
to utilize measurements of the polarization
of dansyl fluorescence to monitor changes
in the oligomeric state. The degree of
polarization provides a measure of a proteins rotational volume and is thus sensitive to self-association (27).
The precipitation curve for the dansyllabeled albumin monomer at a concentration of approximately 2 mglml is given by
the open circles in Fig. 7. The filled circles
are the results of an identical experiment
in which unlabeled albumin was added to
bring the total concentration to 20 mg/ml.
The precipitation curves shown in Fig. 7
were obtained by measurements of the
absorbance at 280 nm. However, measurements of the fluorescence at 500 nm gave
identical curves, indicating that labeling
of albumin with an average of 2.3 mol of
the hydrophobic dansyl chromophore did

a
;i
E
g

80

POLYETHYLENE

GLYCOL

111

not interfere with precipitation by PEG.

Furthermore, the dansyl fluorescence was
strictly proportional to the total concentration of albumin in the supernate, even
when dansyl albumin represented only
about 10% of the total. Thus, dansyl albumin should be useful as a tracer for efficiently monitoring the behavior of albumin in the presence of other plasma proteins.
Also shown in Fig. 7 are the results of
measurements of polarization of the dansyl fluorescence in the supernates. In both
experiments there was only a slight increase in P upon addition of PEG, which
can be attributed to the increased viscosity
of the medium, an effect which probably
also accounts for the slightly higher values
of P at the higher albumin concentration.
Even at concentrations of PEG where most
of the albumin had been precipitated, the
saturated supernates showed no evidence
for self-association. At no time did the
polarization reach even the value of 0.25,
characteristic of the dansyl-labeled dimer
obtained as a by-product during the chromatographic purification. The slight decrease in polarization in the range where
precipitation occurs is explained by the
fact that, even after purification by gel
filtration, the dansyl albumin still contained a small amount of free dye whose
fluorescence was completely depolarized.
Thus, about 3% of the total dansyl fluorescence could not be precipitated, even at
25% PEG.
DISCUSSION

60

i::t \\ 1
II

12

16

A PEG
FIG. 7. Precipitation
of dansyl-labeled
albumin
with PEG-4000 in the presence (0) and absence (0)
of unlabeled albumin (18 mg/ml) in 0.05 M acetate,
0.1 M KCl, pH 4.5. The concentration
of dansyl albumin was approximately
2 mg/ml in both experiments. The upper panel gives the polarization
of
dansyl fluorescence in the supemates, measured at
500 nm with excitation at 340 nm. The temperature
was 25C for the polarization
measurements.

The concentration of PEG required to

precipitate albumin is at a minimum near
the isoelectric point at pH 4.5. At a fixed
concentration of PEG (II%), the pH-solubility profile is extremely sharp and there
is little difference between human and
bovine albumin. The precipitation
is
equally inhibited by increasing concentrations of several different salts, provided
that the pH is at or above the isoelectric
point. At pH 4.0, the precipitation is enhanced by the addition of various salts to
an extent which correlates roughly with
their position in the Hofmeister, or lyotropic, series (28). An exception was the
sulfate anion, which normally occurs at

112

KENNETH

the opposite end of the series. However,

the lyotropic series is based on effects
which are normally expressed at much
higher salt concentrations, suggesting
that a more specific interaction is responsible for the effect of sulfate reported in
Fig. 3.
Edmond and Ogston (31) reported that
PEG and albumin form two immiscible
aqueous phases above certain concentrations. No evidence for the formation of
two liquid phases was found under the
various conditions of the experiments reported here. However, the authors implied
that precipitation of proteins by nonionic
polymers should be considered as a special
case of the same phenomenon in which
one of the phases is a solid solution. One
would then expect the concentration of
protein in the supernate to depend on the
relative volumes of the liquid and the solid
solutions. However, variation of the initial
albumin concentration (and thus the
amount of precipitate formed) had no effect
on the absolute concentrations of albumin
remaining in solution. Furthermore, it
should be pointed out that the binodial
curves characterizing these two-phase systems invariably approach the concentration axes asymptotically (34). Thus, the
concentration of albumin in a saturated
solution should be diminished by even a
slight addition of PEG. As shown in Fig.
4, this was not observed.
In a separate study, it was shown that
the ability of PEG to (a) alter the solubility
of amino acids, (b) perturb the absorption
spectra of aromatic amino acids, and (c)
enhance the fluorescence of l&anilinonaphthalene sulfonate increased steadily
with increasing molecular weight (22).
However, the trend tends to level off at a
molecular weight of about 400, the higher
polymers being only slightly more effective. In contrast, the effectiveness of PEG
as a protein-precipitating agent continues
to increase with an increasing degree of
polymerization, PEG-4000 being substantially more effective than PEG-400 (Figs.
5 and 61, suggesting that the precipitation
mechanism involves additional factors beyond the general perturbing effect of PEG
on the aqueous solvent. The emphasis inthe literature has been on a steric exclu-

C. INGHAM

sion mechanism whereby proteins, upon

addition of PEG, are excluded from domains of the solvent occupied by the network of synthetic polymers (7, 21, 35-37).
The proteins are thus concentrated until
their solubility is exceeded. Presumably,
these steric effects play an increasing role
as the size of PEG and/or the protein is
increased.
It is of interest that, while PEG-4000 is
more effective in terms of the concentration required to effect precipitation, the
400 molecular weight polymer may be
more effective in terms of the selectivity
of precipitation. This is suggested by the
data in Fig. 6, where the separation of the
precipitation curves for albumin monomers and dimers is greater with PEG-400
than with PEG-4000. Honig and Kula (38),
working with yeast extracts, recently concluded that PEG-300 was superior to PEG4000 or PEG-6000 in terms of the separation of a-glucosidase from the remaining
proteins. In view of the greater ease with
which the smaller polymers can be removed from the final product, these observations indicate that the role of the molecular weight of PEG deserves further investigation.
The measurements of the fluorescence
polarization of dansyl-labeled albumin indicate that PEG-induced precipitation is
not preceded by a progressive buildup of
soluble aggregates whose size eventually
exceeds a critical limit. Rather, albumin
remains in a monomeric state in the presence of increasing concentrations of PEG,
right up to the point of precipitation. Thus,
the precipitation process is highly cooperative, involving the simultaneous assembly of large numbers of monomers into
insoluble macroscopic particles. Further
investigations are in progress to ,determine
the effect of PEG on the oligomeric state
of proteins with a known tendency to selfassociate, and these investigations will
evaluate the feasibility of shifting the pfccipitation curves via manipulat;or: ti selfassociation.
ACKNOWLEDGMENTz,
The expert technical assistance of Mr. Ronald
Ling is gratefully
acknowledged.
I also thank MS
Betty Swanson for secretarial
assistance. This re-

PROTEIN

PRECIPITATION

WITH

port represents
Contribution
No. 369 from the
American
Red Cross Blood Research Laboratory,
Bethesda, Maryland.
The work was supported in
part by a Biomedical Research Support Grant (No.
RR 05737) from the National Institutes of Health.
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POLYETHYLENE

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