J. BI OMED. MATER. RES. VOL. 3, PP.

175-189 (1969)
Adsorption of Plasma Proteins in Solution
to Uncharged, Hydrophobic Polymer Surfaces
J . L. BRASH and D. J . LYMAN, Stanford Research Institute, Menlo
Park, California 94025
Summary
Infrared internal reflection spectroscopy has been used to study the adsorption
of certain plasma proteins on a variety of hydrophobic polymer surfaces. The
behavior of the systems studied was almost identical. Under static conditions
the proteins appear to be rapidly adsorbed as monomolecular layers from solu-
tions varying in concentration between a few me-% and normal plasma levels.
These monolayers are deduced to be closely packed arrays in which the protein
molecules appear to retain their native globular form. The bearing of these
results on the mechanism of surface-induced coagulation is significant.
INTRODUCTION
It is of prime interest to elucidate the interaction of synthetic
polymer surfaces with plasma proteins in solution, since these inter-
actions are intimately related to the foreign surface-induced coagula-
tion of blood. Since the intact clotting factors in the cascade
scheme of Maefarlane and others1V2 are proteinaceous and have
physical properties similar to the more abundant plasma proteins,
it is appropriate to draw analogies between the behavior of these two
groups of substances. I n particular it would be illuminating to de-
termine whether a given surface is specific in its interaction with a
particular protein or clotting factor or whether the interaction is
general in the sense of providing an energy source, which can sensitize
all or any of the clotting reactions.
Much previous work has been carried out on the behavior of pro-
teins at surf a~es.~ Most of this has been concerned with the air-
water i nterf a~e,~.~ or the oil-water interface. 3 v 6 Such solid-solution
175
176 J. L. BRASH AND D. J. LYMAN
interfaces as have been studied have been predominantly of the glass
t y ~e. ~- ~ However there is a need for information on protein be-
havior at polymer surfaces. Inasmuch as a relationship has already
been established between surface free energy of uncharged, hydro-
phobic polymers and coagulation time of plasma or blood in contact
with them,lO,ll i t would be diagnostic to further demonstrate a
progressive specific interaction with some blood constituent such as
a protein.
I n this paper, therefore, we report on a variety of these uncharged
hydrophobic surfaces. These surfaces are uncharged in the sense of
having no permanent ionizable groups or semipermanent charges
in the manner of electret materials; they will of course have an
associated zeta potential in contact with a liquid, due to the electrical
double layer. They are also inert in the sense of not containing any
reactive chemical groups such as hydroxyl, isocyanate, etc. They
represent therefore a relatively simple class of materials whose
principal variable property is their surface energy.
Adsorption studies in solution are classically carried out either by
depletion methods in which a highly specific surface area powder is
exposed to the solution and the decrease in concentration of the
latter is measured, or by elution techniques in which the solution
and solid are allowed to react, the complex is isolated, and the
adsorbed species is washed out and measured. The precision of
these methods is dependent on the analytical technique used, and
with presently available tools this is usually not limiting. However
the intrinsic defect of such methods, especially when dealing Kith
biological systems, is that subtle changes such as denaturation at the
surface are not readily detected. It is essential to examine the sub-
strate-adsorbate complex directly. Infrared internal reflection
spectroscopy appears to provide a technique by which this is possible.
The concept of internal reflection spectroscopy was developed by
Fahrenfort12 some years ago, with particular emphasis on the infrared
region, and has been reviewed recently by Harrick.13 The technique
provides a means of obtaining the infrared spectrum of the first few
hundred Angstroms of the surface of a material and therefore could
conceivably be used to detect adsorbed layers. Since the infrared
spectra of proteins and other biological molecules are distinctive and
well documented, the method seemed to have promise for direct
study of the protein-surface complex.
ADSORPTI ON OF PLASMA PROTEI NS 177
EXPERIMENTAL
Materials
The polymer films used were: low-density polyethylene (1-mil film,
Olin Matheson Corp.) ; polystyrene (5-mil foamed sheet, W. R.
Grace Co.)-the foamed material was found more suitable for in-
frared work than conventional clear film because i t is less brittle and
more formable; polydimethyl-siloxane (10-mil medical grade Silastic
film, Dow Corning Corp.) ; and fluorinated ethylene-propylene
copolymer (Teflon FEP 10-mil film, E. I. du Pont Co.) The films
were cleaned by washing in ethanol and then in distilled water.
Detergents were avoided since they adsorb and cannot readily be
completely removed from the surface of the film.
The proteins used were: albumin (4X crystallized, Nutrional Bio-
chemical Co.) ; 7-globulin (Cohn fraction 11, N.B.C.) ; and fibrinogen
(Cohn fraction I, N.B.C.). Albumin and yglobulin were found to
be electrophoretically homogeneous and were used as received.
Fibrinogen was purified by the method of Straughn and Wagner.I4
The Cohn faction was dissolved in a citrate-saline solvent to a con-
centration of about 300 mg-% and precipitated by the addition of
p-alanine. The suspension was equilibrated at ice temperatures for
30 min and then centrifuged at 40C for 25 min at 2OOOg. The
supernatant was discarded and the precipitate redissolved to a con-
centration of -900 mg-% and diluted as required.
Adsorption Procedures
The adsorption of plasma proteins to polymer surfaces was con-
ducted under both static and flow conditions. I n the static experi-
ments, four sections of polymer film (2 x 5 cm) were immersed in
water and protein solution added to make the final correct con-
centration. Care was taken to ensure that the films did not touch
each other or the walls of the beaker. The beaker was covered with
foil (to prevent evaporation) and placed in a 37C water bath. After
the desired time (usually 2 hr) the solution was displaced by a succes-
sion of decantation and dilution steps. The films were then removed
from the beakers, shaken to remove large adhering droplets, and
rinsed by first immersing in 100 ml of distilled water and then holding
in running distilled water for 1 min on each side. Such extensive
178 J. L. BRASH AND D. J. LYMAN
rinsing was necessary to ensure removal of all but adsorbed material.
The films were then placed in a forced-air oven at 50C and dried for
30 min. The infrared internal reflection spectra were then obtained.
I n a few instances, water was removed by lyophilization to ascertain
that exposure to the 50C drying temperatures had no effect on the
adsorbed proteins.
First, the
solution (which was contained in a 1 liter beaker in a 37C water bath)
was pumped through a loop of tubing of the test material using a
tubing pump. Sections of tubing 5 cm long were cut from the loop,
rinsed, and dried as in the static experiments; the sections were slit
lengthwise and the infrared internal reflection spectra taken. This
arrangement was limited to flexible materials available in tube form.
The second procedure, using a Babb-Grimsrud artificial kidney test
ceU,15 allowed polymers in the form of flat sheets to be studied. I n
this arrangement, the polymer films were substituted for the mem-
brane. Again a glass beaker served as reservoir and connections
were made to the cell with Silastic tubing. Rinsing, drying and
spectral measurement were carried out as in the static experiments.
Infrared Internal Reflection Spectra
The spectra were obtained using a Perkin-Elmer 221 spectro-
photometer and a single-beam, internal reflection attachment (Model
9, Wilks Scientific Co.). The optical arrangement is shown sche-
matically in Figure 1. The reflectance cell used 50 X 20 X 1 mm
KRS-5 (thallous bromide-iodide) plates with entrance and exit
faces cut at 45" angles. Spectrometer settings were adjusted to give
I n the flow experiments, two procedures were used.
1
SPECTROPHOTOMETER
I
SAMPLE
SOURCE *
-_2_
-I
KRS5 REFLECTOR PLATE
Fig. 1. Schematic of optical arrangement for infrared reflection spectroscopy.
ADSORPTION OF PLASMA PROTEINS 179
the best resolution consistent with a minimum-noise baseline.
Ordinate expansion was not necessary except in cases where adsorption
was slight. Since conta.ct between film and plate is never complete
and can vary for each experiment, it is not possible to utilize the
same amount of sample area in each run. However, the intensity
of the base polymer spectrum can be used to estimate the relative
degree of contact (i.e., area) by determining the ratio of absorbances
of the band of interest to a suitable internal standard band in the
polymer spectrum. The internal standards used were: the 6.9 p
methylene bending vibration for polyethylene and polystyrene; the
7.1 p methyl bending vibration for polydimethylsiloxane; and the
10.3 p -C-F bending vibration for Teflon FEP.
The KRS-5 reflector plate was cleaned in methanol and polished
with cerium oxide-chromium oxide (slurried in methanol) between
each spectral measurement to remove any protein transferred from
the polymer in the preceding run. However, even with this pro-
cedure complete removal was not possible and a small but significant
layer built up with every spectrum. To compensate for this con-
tamination, a control spectrum was run prior to each adsorbed sample
using the clean, untreated polymer; the difference between the two
absorbance ratios, i.e., the net absorbance ratio, was used in
calculating the results. This ratio can, of course, exhibit occasional
negative values when adsorption is small and accuracy is reduced.
Calibration of the net absorbance ratio in terms of average surface
density of adsorbed material was made by drying measured volumes
of solutions of known concentration on a 100 cm2 area of the polymer
film and measuring the net absorbance ratio for these known surface
densities. This procedure is not suitable for Teflon FEP because its
surface is not wetted by water or by any other convenient protein
solvent. The spraying of droplets of less than lp diameter (from an
atomizer) onto the surface was also ineffective, because they coalesced
before drying was complete. Calibration was obtained by a com-
parative technique, using a previously calibrated surface such as
polyethylene on one side of the reflector plate and Teflon FEP on
the other side. The relative absorbances of the two internal stan-
dard bands (6.9 and 10.3 p ) under identical contact conditions were
thereby obtained and were reproducible within a few per cent.
Assuming that the extinction coefficient of the amide I band of any
protein is independent of the substrate (and this appears to be borne
180 J. L. BRASH AND D. J. LYMAN
out by the results), the calibration of the unknown system was ob-
tained by multiplying the protein calibration on polyethylene by
the ratio of the internal standard adsorbances, e.g., CFEP = Cp~Aa. g/
Alo.z. The method was substantiated by finding that calibration
data obtained directly (where possible) were in good agreement
with those obtained by the comparison technique.
RESULTS AND DISCUSSION
The infrared spectra of proteins have been widely studied.16-19
The principal absorption bands of proteins are the amide A band due
to the N-H group at 3300 em-' and the amide I and amide I1 bands
due to the -CONH- group at 1650 cm-I and 1550 cm-l. These
bands are identical for all proteins and peptides, and this prevents
distinguishing among different proteins on the basis of their infrared
spectra alone. This is illustrated in Figures 2-4, which show the
similar infrared reflection spectra obtained for a polymer surface
exposed to albumin, fibrinogen, and whole blood, respectively. The
latter exposure was made in a flow system,20 which prevents any
blood-air interface and thereby any Langmuir-Blodgett transfer.
The absorption bands for the proteins adsorbed from blood are indis-
tinguishable from those for either albumin or fibrinogen (Figs. 2 and
3)) so it is not possible to say which, if any, of the proteins is preferen-
tially adsorbed.
Since competitive studies cannot be made with this technique,
the approach which must be used is that of a comparative study, in
which each system is investigated independently but under identical
3 4 5 6 7 8
WAVELENGTH - microns
Fig. 2. Infrared reflection spectrum of human serum albumin on polyethylene:
(--) untreated polyethylene; (- - -1 protein treated polyethylene.
ADSORPTION OF PLASMA PROTEINS 181
00
w
a
(L
0
2 0.2
m
0.4
a
06
t
7
I .o
--_ I
co ~ I " ~ ' ' ' ~ ' ~
2 3 4 5 6 7 0 9
WAVELENGTH - microns
Fig. 3. Infrared reflection spectrum of human fibrinogen on Silastic: (-)
untreated Silastic; (- - -) protein treated Silsstic.
2 3 4 5 . 6 7 0 9
WAVELENGTH -microns
Fig. 4. Infrared reflection spectrum of polyethylene exposed to whole blood
(-) untreated polyethylene; (- - -) whole blood treated polyethylene.
conditions. Using this overall approach, each protein-polymer
system was studied by measuring the quantity of protein adsorbed as
a function of solution concentration.
How-
ever, it was found that buffer salts were adsorbed along with the
protein, and that water bound to the buffer could not be removed
by any drying procedure that did not change the protein at the same
time. Such adsorbed water is sufficient to mask the infrared spectra
in the region of interest, i.e., 5-7 p, and cannot be tolerated. It was
necessary, therefore, to conduct the adsorption experiments in dis-
Preliminary work was done using buffered saline at pH 7.4.
182 J . L. BRASH AND D. J . LYMAN
tilled water. (Several additional experiments were conducted at
pH 7.4 using a,n imidazole-HC1 buffer which does not form water-
binding salts. No differences in the results were observed between
this solvent and distilled water.)
The quantity of protein adsorbed on each polymer was deter-
mined from the ratios of their infrared internal reflection bands.
Calibration curves were obtained by depositing known concentrations
of protein on the polymer surface. The absorbance ratios were
linear with surface density) over the range of interest as shown in
Figure 5 for y-globulin on polystyrene. The adsorption behavior
of the polyethylene surface toward three of the more abundant
plasma proteins, fibrinogen, y-globulin, and albumin) is shown in
Figure 6.
The surface concentration of adsorbed protein rapidly reaches a
substantially constant value at solution concentrations of 5-7 mg-yo
and does not change even as solution concentration approaches
normal plasma levels. At very low solution concentrations, there is
a build-up towards this constant) or saturation, value. The scatter
0. I4
rn
D
z
a
m 0.12
i
o!
- 0.10
(d
0
3 0.08
f 0.06
W
..
W
0
LL
0
rn
0.04
+
W
z
0.02
0
I I 1
0 0.2 0.4 0.6 0.8 1.0 1.2 1 . f
SURFACE CONCENTRATION - pgcm' '
Fig. 5. Calibration of the amide I band in the infrared reflection spectrum of
yglobulin on polystyrene.
ADSORPTION OF PLASMA PROTEI NS 183
SOLUTION CONCENTRATION OF PROTEIN - rng. percent
Fig. 6. Adsorption of plasma proteins to polyethylene at 37C. ( A) Fibrinogen;
( 0 ) gamma globulin; (H) albumin.
in the surface concentrations determined at any given solution
concentration reflects the many uncertainties inherent in the method
from a quantitative standpoint.
It can also be seen from Figure 6 that each protein has its own
definite and distinct surface concentration plateau and that the
plateau value increases with molecular weight. It seems reasonable
to interpret these plateaus as corresponding to formation of mono-
layers of protein at the surface. This general behavior has been
observed with other types of macromolecules at the solid-solution
interface2I and is in general typical of all the protein-polymer systems
studies here.
The plateau surface concentrations are given in Table I .
TABLE I
Surface Concentration of Plasma Proteins Adsorbed on Polymer Surfaces
Protein conc, pg/cm2
Polymer Albumin 7-Globulin Fibrinogen
Polystyrene
Polyethylene
Silastic
Teflon FEP
0.5 0.7 1.7
0.8 1 .0 1.3
1.6 1.8 1.6
0.8 0 1.4
184 J. L. BRASH AND D. J. LYMAN
It would appear that, within experimental error, the behavior of a
given protein is similar for all of these surfaces (e.g., fibrinogen). The
most notable exception appears to be r-globulin which has a very
high surface concentration on Silastic and zero surface concentration
on Teflon FEP. The former result may represent a more compact
form of layer on this surface while the latter has no convincing ex-
planation at present. The data did show considerable scatter, and a
few experiments indicated finite adsorption of yglobulin on Teflon
FEP. Albumin also appears to form an unusually compact layer on
Silastic.
The obvious conclusion from these data is that, individually at
least, all proteins behave rather similarly on a wide variety of hydro-
phobic surfaces. It is tempting, therefore, to infer that in a mixture
such as blood the proteins would be adsorbed simply in proportion
to their surface collision frequency or concentration. This suggests
that albumin would be the most abundant protein found at the inter-
face, followed by 7-globulin. This idea is contrary to the findings of
Vroman who has determined that fibrinogen is preferentially ad-
sorbed at tantalum and silicon surfaces in contact with plasma.22
It was also of interest to determine the effect of flow on the mech-
anism of protein adsorption. I n the i n vi vo situation, where a pros-
thetic surface would be exposed to flowing blood, the accompanying
shear stress at the wall might alter adsorption behavior. For
example, adsorption might not occur at all, or i t may be ac-
companied by denaturation, or a continuous adsorption-desorption
process may occur with release into the blood of molecules which
are denatured and activated as a result of contact with the surface.
To study this possibility, we have performed flow experiments on two
of the systems studied under static conditions (namely, Silastic-
albumin and polyethlene-albumin) . The results are summarized in
Table 11. For the Silastic tubing there is no adsorption for up to 12
hr while for Silastic flat sheet a monolayer is almost complete in 15
min, as under static conditions. I n polyethylene, which is not
amenable to study in tube form, monolayering is complete in 5 min
in the flat sheet configuration, again the same as for static conditions.
Thus, adsorption appears to be affected only in the turbulent
region. Where flow is laminar, as in the parallel plate configurations,
adsorption appears to be the same as for static conditions. I n most
areas of the circulation, the critical velocity separating turbulent
ADSORPTION OF PLASMA PROTEINS 185
TABLE II
Flow Systems
Adsorption of Albumin from 20 mg-% Solutions to Polymer Surfaces in
I R net Surface
Surface ml/min number time, min ratio ie/cm2
Flow rate, Reynolds Exposure absorbance cone.,
Silastic:
tubing
(0.7 cm I.D.)
Silastic :
parallel plates
(0.29 cm apart
Polyethylene :
parallel plates
(0.025 cm apart)
Polyethylene
660 2240 240
480
720
960
570 260 15
30
60
240
245 120 5
15
30
60
240
0 1
5
10
15
60
0
0
0.077
0.413
0.066
0.151
0.158
0.200
0.090
0.077
0.071
0.060
0.150
0.110
0.056
0.064
0.081
0.098
0
0
1.0
5.3
0.86
1.97
2.05
2.60
0.75
1.87
1.4
0.7
0.8
1 .0
1.2
and laminar flow is not exceeded; possible exceptions are the aorta
during violent exercise or the regions near heart valves. The study
of adsorption of proteins under static conditions should, therefore,
have valid application to the circulating blood.
An increase in surface concentration beyond monolayering also
occurs at long exposure times in these flow experiments (see Table 11),
for example, after 16 hr in Silastic tubing and after 4 hr in both
Silastic and polyethylene flat sheet. This may not be related to flow
per se but could reflect some change in the protein which results in
adsorption behavior different from that of the native form.
From the surface concentration data in Table I, the protein layer
thickness and the average are perprotein molecule can be calculated,
assuming protein density to be 1.3 and protein molecular weights of
186 J . L. BRASH AND D. J . LYMAN
69 000 for albumin, 160 000 for y-globulin, and 400 000 for fibrinogen.
These calculated values are given in Table 111.
If these experimental values are compared to the values calculated
from the reported dimensionsz3 of the native globular form of the
protein (see Table I V), it should be possible to determine the structure
of the adsorbed layer. The experimental layer thickness values in
TABLE I11
Experimental Dimensions of Protein Layers Adsorbed on Polymer Surfaces
Layer thickness, Average area- per
Protein A molecule, A2
Albumin
7-Globulin
Fibrinogen
Albumin
7-Globulin
Fibrinogen
Albumin
7-Globulin
Fibrinogen
Albumin
7-Globulin
Fibrinogen
on Polystyrene
44
54
130
on Polyethylene
62
77
96
on Silastic
120
138
120
on Teflon FEP
62
0
108
2300
3800
4000
1400
2660
5340
720
1500
4200
1440
0
4760
TABLE IV
Dimensional Data for Plasma Proteins
Diameter, Projected area Length, Projected trea
Protein ii end-on, A 2 ii side-on, A2
Albumin 40 1700 115 4600
7-Globulin 44 2000 235 10300
Fibrinogen 65 4200 475 13000 to
30000
ADSORPTI ON OF PLASMA PROTEI NS 187
Table 111approximate the diameters of the protein molecules, in-
dicating a possible side-on adsorption configuration, whereas the area
data correspond more to a closely packed layer adsorbed on end.
Since the molecular weight data are probably less questionable
than the assumption that the adsorbed protein density is the same as
the crystalline protein density, it is felt that the end-on configuration
should be favored. If anything, the layer density at the surface
would probably be less than that of the crystal and the derived
thickness would be correspondingly greater and more in line with
end-on adsorption.
Attempts to distinguish between these two configuration may well
be unjustified by the accuracy of the data. However, i t does seem
clear that the adsorbed material does not consist of a single layer of
uncoiled protein. Such a layer which in effect would be a two-
dimensional array of amino acids would have a thickness of around
10 A. We conclude then that in general plasma proteins are not
drastically denatured in a dimensional sense at this type of surface,
and if initiation of blood coagulation involves adsorption and activa-
tion by denaturation, then all of these surfaces should be equally
nonthrombogenic, which is not the case.
These data, of course, do not exclude the possibility that the
equilibrium layer is a multilayer of denatured protein, that is, suc-
cessive layers of uncoiled protein stacked one on top of another.
However, if this were the case, i t is difficult to see why the layering
should stop at a definite value greater than one molecule. A more
likely alternative is that the native layer could be preceded by a
layer of denatured protein next to the surface; this would still be
within the limits of the data. However, the layer next to the blood
would be a native layer and there would presumably be no progres-
sive denaturation of protein by contact with this native surface.
Protein adsorption in both static and flow systems is not reversible.
The proteins are not desorbed over a wide range of pH, nor are they
removed by extensive ultrasonic vibration. While this might suggest
that the proteins are uncoiled and strongly bonded to the surface
through multiple contacts or that a surface-denatured layer-native
layer structure exists, such irreversibility has also been observed in
the albumin/glass system in which adsorption in the native state was
indicated.8
It is of interest to comment on the significance of these results in
188 J . L. BRASH AND D. J . LYMAN
regard to the coagulation of blood at polymer surfaces. It would
seem that since factor XI 1 and other clotting factors are themselves
proteins and are similar to the common plasma proteins in physical
properties, then the behavior of these factors at surfaces should also
be similar. Thus factor XI1 is a sialoglycoprotein of molecular
weight around 80 000 and migrates between p- and 7-globulin.
Furthermore the activated form of factor XI 1 behaves as a heavier
macromolecule in the ultracentrifuge than does the intact form ac-
cording to Donaldson and R at n~f f . ~~ The activated form is also
less soluble and i t would seem possible that this could be due to some
conformational change. If this is so, then activation would be
equivalent to a conformational or dimensional change. The con-
clusions from these studies, therefore, tend to refute the contact
activation of factor XI1 as being the initiating step of clotting on
uncharged, hydrophobic polymer surfaces.
The experimental assistance of Miss M. Carini is gratefully acknowledged'
This work was supported by the National Heart Institute, Artificial Heart
program, under Contract PH 43-64-84, and in part by the National I nstititute
of Arthritic and Metabolic Diseases, Artificial Kidney Program, under Contract
PH 43-66-493.
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Received October 11, 1968