- Email: [email protected]
Interaction of fibrinogen with solid surfaces of varying charge and hydrophobic—hydrophilic balance
Interaction of Fibrinogen with Solid Surfaces of Varying Charge and Hydrophobic-HydrophUic Balance II. DynamicExchange between Surface and Solution Molecules J. L. B R A S H , *
S. U N I Y A L , *
C. P U S I N E R I , t AND A. S C H M I T T ~
*Departments of Chemical Engineering and Pathology, McMaster University, Hamilton, Canada, ~Centre de Recherches des Carridres, Rh&ne Poulenc, Saint Fons, France, and $Centre de Recherches sur les Macromolecules, C.N.R.S., Strasbourg, France
Received August 17, 1982; accepted February 3, 1983 In the present work, dynamic aspects of the interaction between fibrinogen and three model polyelectrolyte-complex (PEC) surfaces (one negatively charged, one neutral, and one positively charged) were investigated, the static aspect having been studied previously [Schmitt et al. J. Colloid Interface Sci. 92, 25 (1983)1. The method consists of carrying out preliminary adsorption with radiolabeled fibrinogen and subsequently exposing the surface to a solution of nonlabeled fibrinogen. The radioactivity of the surface is followed continuously as a function of time and, in this way, a precise time dependence of the solution-surface exchange process is obtained. It was shown that in the pseudoplateau region of the isotherm, exchange between solution and interface takes place at two significantly different rates, so that three populations of adsorbed fibrinogen have to be considered: nonexchanging, rapidly exchanging, and slowly exchanging. Lower on the isotherm a single rate of exchange was observed for the negatively charged and neutral surfaces. This rate was similar to that of the slow exchange process occurring at higher concentration. For the positively charged surface, two distinct exchange processes were observed at low as well as at high concentration. No obvious correlation could be found between exchange behavior and the varying characteristics of the different PEC surfaces, namely, charge and hydrophobicity. Taking the present results together with those published previously [Brash et aL Trans. Amer. Soc. Artif. lnt. Organs 20, 69 (1974); Brash and Samak, J. Colloid Interface Sci. 65, 495 (1978)] it is possible to claim as a general rule that the "equilibrium" between an adsorbed protein layer and its solution is only partly reversible, since we find that part of the surface layer is exchangeable and part is not. It is worth noting that this conclusion emerges from experiments conducted over a wide range of time intervals: hours (this work with fibrinogen) versus days [Brash and Samak, J. Colloid Interface Sci. 65, 495 (1978) with albumin]. INTRODUCTION
t i m e scale (i.e., w i t h i n h o u r s o r e v e n days). H o w e v e r , this d o e s n o t m e a n t h a t p r o t e i n m o l e c u l e s r e m a i n a t t a c h e d t o t h e surface, w h a t e v e r t h e b a t h i n g liquid. " C l e a n i n g " o f t h e s u b s t r a t e surface m a y b e a c h i e v e d w i t h a l k a l i n e o r d e t e r g e n t solutions. I n a d d i t i o n , a r a t h e r u n e x p e c t e d finding is t h a t e x c h a n g e can take place between adsorbed proteins a n d p r o t e i n s in solution. T h i s p h e n o m e n o n has b e e n d e a r l y d e m o n s t r a t e d i n experim e n t s r e p o r t e d b y B r a s h et al. (1, 2), u s i n g t h e t e c h n i q u e o f r a d i o a c t i v e labeling. It h a s b e e n suggested t h a t i n t r o d u c t i o n o f t h e labels m a y affect t h e p r o t e i n p r o p e r t i e s , a n d l e a d
P r o t e i n a d s o r p t i o n p l a y s a n i m p o r t a n t role in the c o m p l e x b i o l o g i c a l processes arising w h e n a s y n t h e t i c s u b s t r a t e is b r o u g h t i n t o contact with blood or plasma. The adsorpt i o n process is often d e s c r i b e d as " i r r e v e r s ible," since w h e n c o n t a c t is e s t a b l i s h e d b e t w e e n a solid a d s o r b e n t a n d a p r o t e i n solut i o n , a d s o r p t i o n " e q u i l i b r i u m " is u s u a l l y attained within an hour, and the subsequent r e p l a c e m e n t o f t h e s o l u t i o n b y a p u r e buffer s o l u t i o n u s u a l l y d o e s n o t p r o d u c e a n y significant p r o t e i n d e s o r p t i o n o n a n o r d i n a r y 28 0021-9797/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in arty form reserved.
Journal of Colloid and Interface Science, Vol. 95, No. 1, September 1983
I N T E R A C T I O N S OF F I B R I N O G E N W I T H P O L Y E L E C T R O L Y T E C O M P L E X E S
to an easier desorption (3). Nevertheless, by means of double-labeling experiments Brash and Samak (2) showed that solution molecules adsorb at the same time that surface molecules desorb, proving that the phenomenon is truly an exchange process. In an earlier article, subsequently referred to as Part I (4), we presented adsorption isotherms of fibrinogen on a series of solid surfaces varying in charge and hydrophobic/hydrophilic balance. Analysis of the experimental results showed that there are at least two interracial concentration regimes in these systems. The use of iodine-labeled proteins made it possible to investigate the initial part of the isotherm, and to show that at low bulk concentrations (i.e., C < 10-2 mg ml -~) interactions of fibrinogen molecules with the different surfaces are essentially the same. In contrast, specificity of interactions was apparent in the pseudo-plateau regime, where we postulated an end-on adsorption mechanism. Particularly high surface concentrations were obtained with a positively charged polyelectrolyte complex material of 0.8 mEq g-i ion-exchange capacity. The aim of the present work was to gain additional information concerning the dynamics of fibrinogen interactions with these surfaces. Thus results are presented concerning the extent and rate of self-exchange of fibrinogen on various charged surfaces. The relevance of such an exchange process to blood-material interactions is emphasized by several recent reports. Thus various in vitro (5) and in vivo (6, 7) studies have shown that over relatively brief periods the protein layer adsorbed from blood to various solids varies in composition. These results can only be explained by postulating a dynamic exchange between surface and solution. EXPERIMENTAL
Polymers and Protein The adsorbents used in the present work are polyelectrolyte complexes (designated PECs) prepared by mixing controlled pro-
29
portions of a polyanion (random copolymer of acrylonitrile and sodium methallyl sulfonate) and a polycation (random copolymer of acrylonitrile and 1,2-dimethyl-5-vinylpyridinium iodide). As indicated in Part I (4), the strong attractive electrostatic interactions between the two polyelectrolytes should ensure intimate mixing and prevent any microphase separation. The PEC materials were coated on the inside of 25-cm-long glass tubes of 0.2 cm internal diameter, under dust-free conditions. Experiments were performed with three PECs, of respective stoichiometric ion-exchange capacities, -0.8, 0, and +0.8 mEq g-m of dry polymer. The protein under study was human fibrinogen, purchased from Kabi (Stockholm, Sweden). This material was designated Grade L with clottability greater than 90%. Solutions of 1% (weight percentage) concentration were prepared according to a standard procedure, stored at -20°C, and quickthawed at 37°C just before use (4). Iodine labeling using 1251 (New England Nuclear, Boston) was carried out by the iodine monochloride method. Buffer solutions contained 0.05 M tris(hydroxymethyl)aminomethane, with pH adjusted to 7.35 by addition of a concentrated HC1 solution. Additional details concerning the adsorbents, the determination of protein concentration, the method of labeling, and the way adsorption experiments were performed may be found in Part I (4). Experimental Procedure The method used to study the exchange process is similar to that previously reported by Brash et al. (1, 2). The polymer-coated glass tubes were first equilibrated overnight with buffer solution, then exposed to a labeled fibrinogen solution for 3 hr at a flow rate of 50 ml min -1 (2). After thorough rinsing with the pure buffer solution, the tubes were incorporated into a flow circuit as indicated in Fig. 1. The main improvement with respect to previous work (1, 2) is that Journal of Colloid and Interface Science, Vok 95, No. 1, September 1983
30
BRASH ET AL.
Containing Solution
Reservoir Nonradioactive Fibrinogen
RESULTS
--t ____[
Scintillation Crystal Silastic
~ i
-~-C..... ring Tubing
I
TestSurfacewithAdsorbed Tubing Radioactive Fibrinogen Pump FIG. 1. Schematic of the flow circuit for determining exchange of adsorbed and solution fibrinogen.
the exchange process can be studied virtually in real time since the test surface is placed in the well of the scintillation counter, while nonradioactive protein solution is circulated. A continuous series of 1-min counts is taken during the period of exchange. Thus a virtually continuous record of surface radioactivity can be obtained for determination of exchange rate. In addition all the data refer to a single tube or surface specimen thus avoiding the experimental uncertainty inherent in using a different tube for each experimental time point as was done in previous work (l, 2). The flow rate chosen was 50 ml rain -1, which corresponds for Poiseuille laminar flow to an interfacial shear rate of about 103 sec-% As long as a buffer solution was circulated, the recorded radioactivity remained constant. When this solution was replaced by a nonradioactive protein solution, at a concentration equal to the equilibrium adsorption concentration, a steady decrease in the tube radioactivity was observed. Each experiment lasted from 20 to 24 hr but since this time greatly exceeds the stability period of the fibrinogen solution (4 to 5 hr after which some precipitation becomes evident) the analysis of the exchange data was limited to the first 3 hr. Journal of Colloid and Interface Science, Vol.95, No. 1, September1983
The solution concentrations chosen for study were 10-2 mg m1-1 corresponding to the lower limit of the pseudo plateau region of the isotherm (4), and 0.5 mg ml -~ in the middle of the pseudo-plateau. A low and a high concentration were chosen on the basis that perhaps different exchange behavior would be seen both because the layer structure and the participation of dissolved protein in exchange would be different at low and high concentration. Exchange experiments were run in duplicate at room temperature for each of the three PEC surfaces at each concentration. A first analysis of the experimental data was done assuming an exponential decrease of the surface radioactivity, a quantity directly proportional to the surface concentration of labeled protein F*(t). Such behavior is described by: r*(t) = [r*(0)
-
r*(oo)]
X exp(-kt) + F * ( ~ )
[1]
where F*(0) and F*(oo) are the interracial concentrations of labeled protein at times 0 and infinity and the parameter k-1 represents the relaxation time characterizing the exchange (the time required for a fraction [ 1 - (1/e)] of the exchanging sites to undergo exchange). Table I summarizes the parameter estimates for the various experiments performed. They were obtained using a nonlinear least-squares regression procedure. Parameter p is the percentage exchangeable adsorbed fibrinogen, i.e.:
p=100L
lj
[21
As an illustration, Fig. 2 shows the turnover process observed on the -0.8 PEC surface with a low concentration protein solution (0.01 mg ml-l). We see, by comparing experimental points with the best-fit curve, that the single exponential model appears to be satisfactory in this case.
INTERACTIONS OF FIBRINOGEN WITH POLYELECTROLYTE COMPLEXES TABLE I Parameter Estimates for Exchange o f Adsorbed and Dissolved Fibrinogen on PEC Surfaces ~ PEC interface Solution concentration (rag rnl-~)
0.01
Neutral
+0.8
-0.8
k -~*
pC
k-~
p
k-I
p
27.8 38.5
8 12
90.9 200.0
8 14
100.0 200.0
24 22
36 33
47.6 76.9
25 22
66.7 66.7
35 33
,d
0.50
8.9 12.1
31
the data for the other systems that did not fit well to the single exponential model (marked by asterisk in Table I) were not so obviously indicative of two exchange processes, nonetheless these data were found to give a much better fit to the two-exponential model equation [3] than to equation [ 1]. The parameters characteristic of the two-population model are given in Table II, where the data in the pl and P2 columns correspond to the percentages of each of the exchanging populations: p i = 100 F---L 3
( i = 1,2).
[4]
rj
a A single exponential decay model is assumed (Eq. i=1
[1]). b k-~ is expressed in minutes. ~p is expressed as percentage of surface sites that exchange. d ,, In these runs the fit of data to the model is not satisfactory.
For many of the runs (especially those at high concentration) there are strong indications that the single exponential model (Eq. [1]) is not satisfactory, a result indicated by the asterisk (*) sign in Table I. A striking example is provided by the +0.8 PEC surface, with the high concentration fibrinogen solution (0.5 mg ml-1), as shown in Fig. 3. Initially, we observe a rapid decrease in surface radioactivity, followed by a slower decay. This may be interpreted as showing that more than one exchange process is going on. Attempts were therefore made to fit the data related to the "asterisk-runs" ,with a function involving two relaxation rates: F*(t) = P~ exp(-klt) + I'2 exp(-k2t) + r3
[3]
in which the first and second termsdescribe the turnover of two different populations of bound molecules. The solid line in Fig. 3 represents the best fit of data points to this double exponential model and as can be seen, the fit is excellent over the entire 3-hr time interval of the experiment. Although plots of
DISCUSSION
A first observation concerns experimental precision. From the data in Tables I and II, it is seen that the duplicate values of the exchange parameters are rather widely dispersed, especially those of the relaxation times, k -1. The considerable improvement in quantity and quality of data achieved with our modified experimental technique might have been expected to yield better reproducibility. Similar data scattering was observed in Part I (4), for the adsorption isotherms on the PEC interfaces. We have no explanation to propose for this disappointing behavior. It should be pointed out, however, that great care was taken in the preparation of the PEC interfaces and in the performance of the experiments. Moreover the technique itself is not inherently "noisy" since better reproducibility has been obtained with other surfaces. A common feature emerges from the parameter estimates of Tables I and II: all surfaces display a significant population of nonexchanging molecules, and this population is proportionately higher for the low solution concentration (10 -2 mg mFl). This observation is in agreement with the arguments developed in Part I (4), where we suggested that in the low concentration regime, molecules are strongly bound to the interfaces Journal of Colloid and Interface Science, Vol. 95, No. 1, September 1983
32
BRASH ET AL. 100
90
80
(.9 Z Z H
70
ILl
rr
6O
M ~-
5O
O H O 0C I-'Z W W 0.
20
I0
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
10
20
30
40
59
gO
70
80
90
100
1.1.0
1~0
130
140
150
1G0
170
180
TIME( MIN )
FIG. 2. Solution-surface exchange of fibrinogen on -0.8 PEC at 0.01 mg ml-l: experimental points (.) and best fit ( ).
(side on adsorption) and would be less easily exchanged, thus leading to relatively low values of p and relatively high values of k -1. For the neutral and negatively charged interfaces at low concentration, the fit to a single exponential model was satisfactory, implying only one population of exchanging protein molecules. In these systems the exchange process is slow, with a relaxation time of the order of 100 to 200 min. For all the other conditions (experiments indicated by asterisk in Table I), two exchanging populations (rapidly exchanging and slowly exchanging) had to be considered. However, the slowly exchanging populations again show relaxation times of 100 to 200 min. (The +0.8 PEC at high concentration exhibits unique behavior and is discussed separately.) This observation suggests that the Journal of Colloid and Interface Science, V o l . 9 5 , N o . 1, S e p t e m b e r
1983
slowly exchanging populations have similar binding characteristics. Furthermore for the neutral and negative surfaces it appears that only the slowly exchanging sites are filled at low concentration whereas slow and fast are occupied at high concentration. The data of Table II show that, with the exception of the +0.8 PEC at high concentration, the percentage of rapidly-exchanging proteins (parameter Pl) remains small (less than 6%) although significant. These rapidly exchanging proteins may represent loosely attached molecules. There are several possible reasons for differences in strength of attachment. For example, there could be strong and weak binding sites on both the protein and the surface. Alternatively, the proteins could exhibit varying numbers of surface attachments, as has been pointed out by others,
33
INTERACTIONS OF FIBRINOGEN WITH POLYELECTROLYTE COMPLEXES 100
$0
80 Z I-4 Z H
70 •
-r I.J.J e,." >..
H I-(J E O H O E Z lad ~ ¢Y W 0.
°•
. ** ~
I 40
I 50
°°°o°.° ° °o• • * •°*°*°° *°,.
, %
°
• °%° °
%•. °.o ° * f ° ° ° %
*
o °*• °
°% ° °
60
50
40
3O
20
10
I 10
I 20
I 30
I 80
I 70
I 80
I S0
I 100
I 110
I 120
I 130
I 140
I 150
I 160
I 170
180
TIME( MIN )
FIG. 3. Solution-surface exchange o f fibrinogen on +0.8 PEC at 0.5 m g ml-~: experimental points ( . ) a n d best fit ( ).
for both protein (8) and synthetic polymer adsorption (9). The short relaxation times observed ('-, 100 sec) should be interpreted with caution, since the process may well be
partly diffusion controlled. This possibility can be explored as follows: the mean time necessary for a desorbed molecule to diffuse a small distance 6 from the tube wall is:
T A B L E II Parameter Estimates from Fitting Exchange Data to Eq. Solution concentration (rag ml -I)
0.01
0.50
+0.8 k71
pl
[3] a
Neutral k21
.o2
kT I
Px
k~ 1
-0.8 -°2
k71
Pl
--
9.1 b
--
200.0 b
24 22
100.0 90.9
33 34
2.6 0.7
4 2
125 40.0
11 7
--
--
90.9 b
~
--
200.0 b
8 14
---
5.9 4.4
31 24
500.0 277.8
26 28
7.7 3.1
4 6
166.7 83.3
24 23
10.0 4.0
6 4
k21
P2
a k i '1s are expressed in minutes; p~ a n d P2, the percentages of rapidly a n d slowly exchanging populations, are defined in Eq. [4]. b In these runs the parameters are for fit o f data to Eq. [1].
Journal of Colloid and Interface Science, Vol. 95, No. 1, September 1983
34
B R A S H E T AL.
"rD "~ b2/2D
[5]
where D is the diffusion coefficient of the protein. On the other hand, the convective velocity at this point is: vc = Gf
[6]
G being the interfacial shear rate. This leads to a mean convective time rc --- l/2Gf necessary to leave the capillary of length l. Thus, the total time to escape the field of investigation is r = rc + rD. Minimizing this time with respect to 6 gives:
( ID ~1/3 fN~k~]
;
3 62 r~i,-~D.
[71
The thickness fN characterizes the so-called Nernst diffusion layer. Using appropriate numerical values (I = 5 cm; G = 103 sec-l; D ~ 1 0 - 7 c m 2 sec -1) we obtain: fin --- 6.3 #m;
"rmin --~
6 sec.
[8]
When compared to the mean relaxation time of 100 sec, r~in is small but not negligible. The unique behavior of the +0.8 PEC material at high concentration requires additional discussion. First, the percentage of fast exchanging molecules (k-~ - 300 sec) is relatively high. It is pertinent here to refer to the discussion in Part I (4), where we noted that the high P values observed for this surface at high concentration are in fact greater than would be expected for a close-packed end-on monolayer. If the additional adsorption represents a "second" layer, then molecules in this second layer would have relatively small adsorption energies. Second, the slow exchange process in this system is considerably slower than for the other experimental conditions (400 min relaxation time compared to 100 to 200 min). A tentative explanation is that this second (slowly exchanging) population of molecules, in order to desorb, has to cross the barrier of the rapidly exchanging surface population which is numerically greater than for the other systems. This requirement would give rise to two markedly different relaxation times as is Journal of Colloid and Interface Science. Vol. 95, No. 1, September 1983
evident in Fig. 3. It would also provide a possible explanation for the different characteristics of the "slow" exchanging populations on the +0.8 PEC at 0.5 mg m1-1 versus the other conditions. Other authors have described behavior which they interpreted as showing the existence of several "populations" of adsorbed molecules (10-12). The evidence in support of such interpretations is usually the observation of partial desorption in response to various treatments of the adsorbed protein. For example, Beissinger and Leonard (10) found that 3,-globulin adsorbed on quartz could be partially desorbed by rinsing with buffer. Dillman and Miller (11) concluded that adsorption of various proteins on neutral and negatively charged surfaces takes place in two distinct ways, distinguished by whether or not the protein is removed by 0.1 N NaOH. Very recently, Robertson et al. (12) showed via exchange experiments using fluorescently labeled proteins that for both albumin and fibrinogen adsorbed on silicone rubber, there are reversibly and irreversibly adsorbed populations. In the discussion thus far, we have not considered the two key parameters characterizing the different interfaces, namely, their net electrical fixed charge and their hydrophobicity (related to swelling in water). Apparently, as was found for the adsorption isotherms (4) none of these parameters has a strong influence on self exchange. If we consider the total percentage exchangeable fibrinogen (denoted pX = p~ + P2), w e see that at low concentration, it is the -0.8 PEC which displays the highest pX value, about twice that for the other surfaces. Since this surface is the most hydrophilic and its net electrical charge is of the same sign as the fibrinogen molecule at pH 7.35, it may be that detachment of an adsorbed molecule is relatively easy. At high concentration, this argument apparently does not hold and we observe the sequence: pZ+o.8> pT-o8 > p~e,~
INTERACTIONS OF FIBRINOGEN WITH POLYELECTROLYTECOMPLEXES
35
to be compared with the sequence of F values in the same concentration regime (4):
so that the rate constant k associated with this model is:
/~+0.8 > ~-0.8 ~> Fneut.
k = ]~eCB .
If the present results are compared with those of Brash and Samak (2), the main conclusion is that exchange is fast in the present systems compared to the polyethylene/albumin system studied by these authors. The turnover of fibrinogen adsorbed on glass surfaces (13) is more akin to the present systems in terms of quantities and rates. This is not surprising since as already noted some similarity exists in the adsorption behavior. Finally, if we consider the mechanism of the protein turnover, the present work leads essentially to the same conclusions as those of Brash and Samak. Two schemes can be postulated to describe exchange between surface and solution. The related chemical equations are as follows:
However, because of the poor reproducibility of data from one experiment to another, it was not possible precisely to check the validity of Eq. [ 12] in the high concentration (isotherm plateau) regime, where CA + CA* (and r = (CA + CA*) A, A being the thickness of the layer) remains almost constant with change in cB. One may conclude from the present set of experiments that for bulky molecules, the exchange between interface and solution is probably more complex than implied by Eq. [9]. Two observations point clearly to this conclusion: (i) we often had to consider the existence of three adsorbed populations in the data analysis and (ii) the rate of the slow exchange process does not depend on bulk concentration, with one exception where the variation is opposite to what Eq. [12] predicts. More experimental data are obviously required if the mechanisms are to be described in detail.
B+A* ~B*+A
[9]
L and
/~ A* ~ B* A ~ B
[10]
where A and B refer to adsorbed and bulk molecules, respectively, with the asterisk denoting radiolabeled molecules. Equations [10] describe an isomerization mechanism, that is, an exchange across a potential barrier. It should be noted that via the structure of the adsorbed layer, rate constants /q could depend indirectly on the concentration of the bathing solution, although proportionality is not expected. Therefore, the fact that no desorption is observed in the presence of pure buffer solution, a result quite general with hydrophobic surfaces (2, 3, 14, 15) strongly indicates that this model is not appropriate to describe exchange. If one works under quasi-open system conditions as in the present work, with cB, - 0, the chemical flux related to Eq. [9] is given by the equation:
dCA, = /~eCBCA* dt
[ 11 ]
[12]
SUMMARY In the present work, dynamic aspects of the interaction between fibrinogen and model polyelectrolyte-complex (PEC) surfaces was investigated, the static aspect having been studied previously (4). The method used was similar to that developed previously by Brash and co-workers (1, 2). It consists of carrying out preliminary adsorption with radiolabeled proteins and subsequently exposing the surface to a solution of nonlabeled molecules. The radioactivity of the surface is followed continuously as a function of time and, in this way, a precise time-dependence of the related processes can be obtained. This technique enabled us to show that in the pseudo-plateau region of the isotherm, exchange between solution and interface takes place at two significantly different rates, so that three populations of adsorbed fibrinogen have to be considered: nonexchanging, rapidly exchanging (small percentage, except Journal of Colloid and Interface Science,
Vol. 95, No. 1, Sep~ernber / 983
36
BRASH ET AL.
for the positively charged PEC surface), and slowly exchanging. In the lower concentration region, the neutral and negative PECs show only one, relatively slow, exchange process (two surface populations) whereas the positive PEC again shows a fast and a slow exchange process. Unique behavior of the positive PEC was also observed with regard to its adsorption isotherm (4). Thus, taking the present results together with those published previously (1, 2) it is possible to claim as a general rule that the "equilibrium" between an adsorbed protein layer and its solution is only partly reversible, since we find that part of the surface layer is exchangeable and part is not. It is worth noting that this conclusion emerges from experiments conducted over much different time intervals: hours (this work with fibrinogen) versus days (Brash et al., with albumin). Some interesting comparisons can be made with previous studies of the same systems (4). In particular, the positively charged PEC interface displays unique behavior from both static and dynamic points of view. To explain the high values of interfacial concentration on this surface, we proposed the existence of a second layer consisting of loosely attached molecules. This seems to be confirmed by the exchange results, where the percentage of fast exchanging proteins is considerably greater than for other surfaces. Finally, it should be mentioned that no obvious correlation could be found between exchange behavior and the varying characteristics of the different PEC surfaces, namely, charge and hydrophobicity.
Journal of Colloid and Interface Science. Vol. 95, NO. 1, September 1983
ACKNOWLEDGMENTS The authors are grateful for financial support of this work by the Medical Research Council of Canada, the Ontario Heart Foundation, and the Centre National de la Recherche Scientifique (France).
REFERENCES 1. Brash, J. L., Uniyal, S., and Samak, Q., Trans. Amer. Soc. Artif Int. Organs 20, 69 (1974). 2. Brash, J. L., and Samak, Q. M., J. Colloid Interface Sci. 65, 495 (1978). 3. Norde, W., in "Adhesion and Adsorption of Polymers" (L. H. Lee, Ed.), p. 801. Plenum, New York, 1980. 4. Schmitt, A., Varoqui, R., Uniyal, S., Brash, J. L., and Pusineri, C., J. Colloid Interface Sci. 92, 25 (1983). 5. Vroman, L., Adams, A. L., Fischer, G. C., and Munoz, P. C., Blood55, 156 (1980). 6. Basse-Cathalinat, B., Baquey, C., Llabrador, Y., and Fleury, A., Int. J. Appl. Rad. Isot. 31, 747 (1980). 7. Ihlenfeld, J. V., and Cooper, S. L., J. Biomed. Mater. Res. 13, 577 (1979). 8. Morrissey, B. W., and Stromberg, R. R., J. Colloid Interface Sci. 46, 152 (1974). 9. Grant, W. H., Morrissey, B. W., and Stromberg, R. R., in "Adhesion Science and Technology" (L. H. Lee, Ed.), Vol. 9A, pp. 43-54. Plenum Press, New York, 1975. 10. Beissinger, R. L., and Leonard, E. F., ASA[O J. 3, 160 (1980). 11. Dillman, W. J., and Miller, I. F., J. Colloidlnterface Sci. 44, 221 (1973). 12. Lok, B. K., Cheng, Y. L., and Robertson, C. R., J. Colloid Interface Sci. 91, 104 (1983). 13. Chan, B. M. C., and Brash, J. L., J. Colloid Interface Sci. 82, 217 (1981). 14. Brash, J. L., and Lyman, D. J., J. Biomed. Mater. Res. 3, 175 (1969). 15. Morrissey, B. W., Ann. iV. Y. Acad. ScL 283, 50 (1977).
S. U N I Y A L , *
C. P U S I N E R I , t AND A. S C H M I T T ~
*Departments of Chemical Engineering and Pathology, McMaster University, Hamilton, Canada, ~Centre de Recherches des Carridres, Rh&ne Poulenc, Saint Fons, France, and $Centre de Recherches sur les Macromolecules, C.N.R.S., Strasbourg, France
Received August 17, 1982; accepted February 3, 1983 In the present work, dynamic aspects of the interaction between fibrinogen and three model polyelectrolyte-complex (PEC) surfaces (one negatively charged, one neutral, and one positively charged) were investigated, the static aspect having been studied previously [Schmitt et al. J. Colloid Interface Sci. 92, 25 (1983)1. The method consists of carrying out preliminary adsorption with radiolabeled fibrinogen and subsequently exposing the surface to a solution of nonlabeled fibrinogen. The radioactivity of the surface is followed continuously as a function of time and, in this way, a precise time dependence of the solution-surface exchange process is obtained. It was shown that in the pseudoplateau region of the isotherm, exchange between solution and interface takes place at two significantly different rates, so that three populations of adsorbed fibrinogen have to be considered: nonexchanging, rapidly exchanging, and slowly exchanging. Lower on the isotherm a single rate of exchange was observed for the negatively charged and neutral surfaces. This rate was similar to that of the slow exchange process occurring at higher concentration. For the positively charged surface, two distinct exchange processes were observed at low as well as at high concentration. No obvious correlation could be found between exchange behavior and the varying characteristics of the different PEC surfaces, namely, charge and hydrophobicity. Taking the present results together with those published previously [Brash et aL Trans. Amer. Soc. Artif. lnt. Organs 20, 69 (1974); Brash and Samak, J. Colloid Interface Sci. 65, 495 (1978)] it is possible to claim as a general rule that the "equilibrium" between an adsorbed protein layer and its solution is only partly reversible, since we find that part of the surface layer is exchangeable and part is not. It is worth noting that this conclusion emerges from experiments conducted over a wide range of time intervals: hours (this work with fibrinogen) versus days [Brash and Samak, J. Colloid Interface Sci. 65, 495 (1978) with albumin]. INTRODUCTION
t i m e scale (i.e., w i t h i n h o u r s o r e v e n days). H o w e v e r , this d o e s n o t m e a n t h a t p r o t e i n m o l e c u l e s r e m a i n a t t a c h e d t o t h e surface, w h a t e v e r t h e b a t h i n g liquid. " C l e a n i n g " o f t h e s u b s t r a t e surface m a y b e a c h i e v e d w i t h a l k a l i n e o r d e t e r g e n t solutions. I n a d d i t i o n , a r a t h e r u n e x p e c t e d finding is t h a t e x c h a n g e can take place between adsorbed proteins a n d p r o t e i n s in solution. T h i s p h e n o m e n o n has b e e n d e a r l y d e m o n s t r a t e d i n experim e n t s r e p o r t e d b y B r a s h et al. (1, 2), u s i n g t h e t e c h n i q u e o f r a d i o a c t i v e labeling. It h a s b e e n suggested t h a t i n t r o d u c t i o n o f t h e labels m a y affect t h e p r o t e i n p r o p e r t i e s , a n d l e a d
P r o t e i n a d s o r p t i o n p l a y s a n i m p o r t a n t role in the c o m p l e x b i o l o g i c a l processes arising w h e n a s y n t h e t i c s u b s t r a t e is b r o u g h t i n t o contact with blood or plasma. The adsorpt i o n process is often d e s c r i b e d as " i r r e v e r s ible," since w h e n c o n t a c t is e s t a b l i s h e d b e t w e e n a solid a d s o r b e n t a n d a p r o t e i n solut i o n , a d s o r p t i o n " e q u i l i b r i u m " is u s u a l l y attained within an hour, and the subsequent r e p l a c e m e n t o f t h e s o l u t i o n b y a p u r e buffer s o l u t i o n u s u a l l y d o e s n o t p r o d u c e a n y significant p r o t e i n d e s o r p t i o n o n a n o r d i n a r y 28 0021-9797/83 $3.00 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in arty form reserved.
Journal of Colloid and Interface Science, Vol. 95, No. 1, September 1983
I N T E R A C T I O N S OF F I B R I N O G E N W I T H P O L Y E L E C T R O L Y T E C O M P L E X E S
to an easier desorption (3). Nevertheless, by means of double-labeling experiments Brash and Samak (2) showed that solution molecules adsorb at the same time that surface molecules desorb, proving that the phenomenon is truly an exchange process. In an earlier article, subsequently referred to as Part I (4), we presented adsorption isotherms of fibrinogen on a series of solid surfaces varying in charge and hydrophobic/hydrophilic balance. Analysis of the experimental results showed that there are at least two interracial concentration regimes in these systems. The use of iodine-labeled proteins made it possible to investigate the initial part of the isotherm, and to show that at low bulk concentrations (i.e., C < 10-2 mg ml -~) interactions of fibrinogen molecules with the different surfaces are essentially the same. In contrast, specificity of interactions was apparent in the pseudo-plateau regime, where we postulated an end-on adsorption mechanism. Particularly high surface concentrations were obtained with a positively charged polyelectrolyte complex material of 0.8 mEq g-i ion-exchange capacity. The aim of the present work was to gain additional information concerning the dynamics of fibrinogen interactions with these surfaces. Thus results are presented concerning the extent and rate of self-exchange of fibrinogen on various charged surfaces. The relevance of such an exchange process to blood-material interactions is emphasized by several recent reports. Thus various in vitro (5) and in vivo (6, 7) studies have shown that over relatively brief periods the protein layer adsorbed from blood to various solids varies in composition. These results can only be explained by postulating a dynamic exchange between surface and solution. EXPERIMENTAL
Polymers and Protein The adsorbents used in the present work are polyelectrolyte complexes (designated PECs) prepared by mixing controlled pro-
29
portions of a polyanion (random copolymer of acrylonitrile and sodium methallyl sulfonate) and a polycation (random copolymer of acrylonitrile and 1,2-dimethyl-5-vinylpyridinium iodide). As indicated in Part I (4), the strong attractive electrostatic interactions between the two polyelectrolytes should ensure intimate mixing and prevent any microphase separation. The PEC materials were coated on the inside of 25-cm-long glass tubes of 0.2 cm internal diameter, under dust-free conditions. Experiments were performed with three PECs, of respective stoichiometric ion-exchange capacities, -0.8, 0, and +0.8 mEq g-m of dry polymer. The protein under study was human fibrinogen, purchased from Kabi (Stockholm, Sweden). This material was designated Grade L with clottability greater than 90%. Solutions of 1% (weight percentage) concentration were prepared according to a standard procedure, stored at -20°C, and quickthawed at 37°C just before use (4). Iodine labeling using 1251 (New England Nuclear, Boston) was carried out by the iodine monochloride method. Buffer solutions contained 0.05 M tris(hydroxymethyl)aminomethane, with pH adjusted to 7.35 by addition of a concentrated HC1 solution. Additional details concerning the adsorbents, the determination of protein concentration, the method of labeling, and the way adsorption experiments were performed may be found in Part I (4). Experimental Procedure The method used to study the exchange process is similar to that previously reported by Brash et al. (1, 2). The polymer-coated glass tubes were first equilibrated overnight with buffer solution, then exposed to a labeled fibrinogen solution for 3 hr at a flow rate of 50 ml min -1 (2). After thorough rinsing with the pure buffer solution, the tubes were incorporated into a flow circuit as indicated in Fig. 1. The main improvement with respect to previous work (1, 2) is that Journal of Colloid and Interface Science, Vok 95, No. 1, September 1983
30
BRASH ET AL.
Containing Solution
Reservoir Nonradioactive Fibrinogen
RESULTS
--t ____[
Scintillation Crystal Silastic
~ i
-~-C..... ring Tubing
I
TestSurfacewithAdsorbed Tubing Radioactive Fibrinogen Pump FIG. 1. Schematic of the flow circuit for determining exchange of adsorbed and solution fibrinogen.
the exchange process can be studied virtually in real time since the test surface is placed in the well of the scintillation counter, while nonradioactive protein solution is circulated. A continuous series of 1-min counts is taken during the period of exchange. Thus a virtually continuous record of surface radioactivity can be obtained for determination of exchange rate. In addition all the data refer to a single tube or surface specimen thus avoiding the experimental uncertainty inherent in using a different tube for each experimental time point as was done in previous work (l, 2). The flow rate chosen was 50 ml rain -1, which corresponds for Poiseuille laminar flow to an interfacial shear rate of about 103 sec-% As long as a buffer solution was circulated, the recorded radioactivity remained constant. When this solution was replaced by a nonradioactive protein solution, at a concentration equal to the equilibrium adsorption concentration, a steady decrease in the tube radioactivity was observed. Each experiment lasted from 20 to 24 hr but since this time greatly exceeds the stability period of the fibrinogen solution (4 to 5 hr after which some precipitation becomes evident) the analysis of the exchange data was limited to the first 3 hr. Journal of Colloid and Interface Science, Vol.95, No. 1, September1983
The solution concentrations chosen for study were 10-2 mg m1-1 corresponding to the lower limit of the pseudo plateau region of the isotherm (4), and 0.5 mg ml -~ in the middle of the pseudo-plateau. A low and a high concentration were chosen on the basis that perhaps different exchange behavior would be seen both because the layer structure and the participation of dissolved protein in exchange would be different at low and high concentration. Exchange experiments were run in duplicate at room temperature for each of the three PEC surfaces at each concentration. A first analysis of the experimental data was done assuming an exponential decrease of the surface radioactivity, a quantity directly proportional to the surface concentration of labeled protein F*(t). Such behavior is described by: r*(t) = [r*(0)
-
r*(oo)]
X exp(-kt) + F * ( ~ )
[1]
where F*(0) and F*(oo) are the interracial concentrations of labeled protein at times 0 and infinity and the parameter k-1 represents the relaxation time characterizing the exchange (the time required for a fraction [ 1 - (1/e)] of the exchanging sites to undergo exchange). Table I summarizes the parameter estimates for the various experiments performed. They were obtained using a nonlinear least-squares regression procedure. Parameter p is the percentage exchangeable adsorbed fibrinogen, i.e.:
p=100L
lj
[21
As an illustration, Fig. 2 shows the turnover process observed on the -0.8 PEC surface with a low concentration protein solution (0.01 mg ml-l). We see, by comparing experimental points with the best-fit curve, that the single exponential model appears to be satisfactory in this case.
INTERACTIONS OF FIBRINOGEN WITH POLYELECTROLYTE COMPLEXES TABLE I Parameter Estimates for Exchange o f Adsorbed and Dissolved Fibrinogen on PEC Surfaces ~ PEC interface Solution concentration (rag rnl-~)
0.01
Neutral
+0.8
-0.8
k -~*
pC
k-~
p
k-I
p
27.8 38.5
8 12
90.9 200.0
8 14
100.0 200.0
24 22
36 33
47.6 76.9
25 22
66.7 66.7
35 33
,d
0.50
8.9 12.1
31
the data for the other systems that did not fit well to the single exponential model (marked by asterisk in Table I) were not so obviously indicative of two exchange processes, nonetheless these data were found to give a much better fit to the two-exponential model equation [3] than to equation [ 1]. The parameters characteristic of the two-population model are given in Table II, where the data in the pl and P2 columns correspond to the percentages of each of the exchanging populations: p i = 100 F---L 3
( i = 1,2).
[4]
rj
a A single exponential decay model is assumed (Eq. i=1
[1]). b k-~ is expressed in minutes. ~p is expressed as percentage of surface sites that exchange. d ,, In these runs the fit of data to the model is not satisfactory.
For many of the runs (especially those at high concentration) there are strong indications that the single exponential model (Eq. [1]) is not satisfactory, a result indicated by the asterisk (*) sign in Table I. A striking example is provided by the +0.8 PEC surface, with the high concentration fibrinogen solution (0.5 mg ml-1), as shown in Fig. 3. Initially, we observe a rapid decrease in surface radioactivity, followed by a slower decay. This may be interpreted as showing that more than one exchange process is going on. Attempts were therefore made to fit the data related to the "asterisk-runs" ,with a function involving two relaxation rates: F*(t) = P~ exp(-klt) + I'2 exp(-k2t) + r3
[3]
in which the first and second termsdescribe the turnover of two different populations of bound molecules. The solid line in Fig. 3 represents the best fit of data points to this double exponential model and as can be seen, the fit is excellent over the entire 3-hr time interval of the experiment. Although plots of
DISCUSSION
A first observation concerns experimental precision. From the data in Tables I and II, it is seen that the duplicate values of the exchange parameters are rather widely dispersed, especially those of the relaxation times, k -1. The considerable improvement in quantity and quality of data achieved with our modified experimental technique might have been expected to yield better reproducibility. Similar data scattering was observed in Part I (4), for the adsorption isotherms on the PEC interfaces. We have no explanation to propose for this disappointing behavior. It should be pointed out, however, that great care was taken in the preparation of the PEC interfaces and in the performance of the experiments. Moreover the technique itself is not inherently "noisy" since better reproducibility has been obtained with other surfaces. A common feature emerges from the parameter estimates of Tables I and II: all surfaces display a significant population of nonexchanging molecules, and this population is proportionately higher for the low solution concentration (10 -2 mg mFl). This observation is in agreement with the arguments developed in Part I (4), where we suggested that in the low concentration regime, molecules are strongly bound to the interfaces Journal of Colloid and Interface Science, Vol. 95, No. 1, September 1983
32
BRASH ET AL. 100
90
80
(.9 Z Z H
70
ILl
rr
6O
M ~-
5O
O H O 0C I-'Z W W 0.
20
I0
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
10
20
30
40
59
gO
70
80
90
100
1.1.0
1~0
130
140
150
1G0
170
180
TIME( MIN )
FIG. 2. Solution-surface exchange of fibrinogen on -0.8 PEC at 0.01 mg ml-l: experimental points (.) and best fit ( ).
(side on adsorption) and would be less easily exchanged, thus leading to relatively low values of p and relatively high values of k -1. For the neutral and negatively charged interfaces at low concentration, the fit to a single exponential model was satisfactory, implying only one population of exchanging protein molecules. In these systems the exchange process is slow, with a relaxation time of the order of 100 to 200 min. For all the other conditions (experiments indicated by asterisk in Table I), two exchanging populations (rapidly exchanging and slowly exchanging) had to be considered. However, the slowly exchanging populations again show relaxation times of 100 to 200 min. (The +0.8 PEC at high concentration exhibits unique behavior and is discussed separately.) This observation suggests that the Journal of Colloid and Interface Science, V o l . 9 5 , N o . 1, S e p t e m b e r
1983
slowly exchanging populations have similar binding characteristics. Furthermore for the neutral and negative surfaces it appears that only the slowly exchanging sites are filled at low concentration whereas slow and fast are occupied at high concentration. The data of Table II show that, with the exception of the +0.8 PEC at high concentration, the percentage of rapidly-exchanging proteins (parameter Pl) remains small (less than 6%) although significant. These rapidly exchanging proteins may represent loosely attached molecules. There are several possible reasons for differences in strength of attachment. For example, there could be strong and weak binding sites on both the protein and the surface. Alternatively, the proteins could exhibit varying numbers of surface attachments, as has been pointed out by others,
33
INTERACTIONS OF FIBRINOGEN WITH POLYELECTROLYTE COMPLEXES 100
$0
80 Z I-4 Z H
70 •
-r I.J.J e,." >..
H I-(J E O H O E Z lad ~ ¢Y W 0.
°•
. ** ~
I 40
I 50
°°°o°.° ° °o• • * •°*°*°° *°,.
, %
°
• °%° °
%•. °.o ° * f ° ° ° %
*
o °*• °
°% ° °
60
50
40
3O
20
10
I 10
I 20
I 30
I 80
I 70
I 80
I S0
I 100
I 110
I 120
I 130
I 140
I 150
I 160
I 170
180
TIME( MIN )
FIG. 3. Solution-surface exchange o f fibrinogen on +0.8 PEC at 0.5 m g ml-~: experimental points ( . ) a n d best fit ( ).
for both protein (8) and synthetic polymer adsorption (9). The short relaxation times observed ('-, 100 sec) should be interpreted with caution, since the process may well be
partly diffusion controlled. This possibility can be explored as follows: the mean time necessary for a desorbed molecule to diffuse a small distance 6 from the tube wall is:
T A B L E II Parameter Estimates from Fitting Exchange Data to Eq. Solution concentration (rag ml -I)
0.01
0.50
+0.8 k71
pl
[3] a
Neutral k21
.o2
kT I
Px
k~ 1
-0.8 -°2
k71
Pl
--
9.1 b
--
200.0 b
24 22
100.0 90.9
33 34
2.6 0.7
4 2
125 40.0
11 7
--
--
90.9 b
~
--
200.0 b
8 14
---
5.9 4.4
31 24
500.0 277.8
26 28
7.7 3.1
4 6
166.7 83.3
24 23
10.0 4.0
6 4
k21
P2
a k i '1s are expressed in minutes; p~ a n d P2, the percentages of rapidly a n d slowly exchanging populations, are defined in Eq. [4]. b In these runs the parameters are for fit o f data to Eq. [1].
Journal of Colloid and Interface Science, Vol. 95, No. 1, September 1983
34
B R A S H E T AL.
"rD "~ b2/2D
[5]
where D is the diffusion coefficient of the protein. On the other hand, the convective velocity at this point is: vc = Gf
[6]
G being the interfacial shear rate. This leads to a mean convective time rc --- l/2Gf necessary to leave the capillary of length l. Thus, the total time to escape the field of investigation is r = rc + rD. Minimizing this time with respect to 6 gives:
( ID ~1/3 fN~k~]
;
3 62 r~i,-~D.
[71
The thickness fN characterizes the so-called Nernst diffusion layer. Using appropriate numerical values (I = 5 cm; G = 103 sec-l; D ~ 1 0 - 7 c m 2 sec -1) we obtain: fin --- 6.3 #m;
"rmin --~
6 sec.
[8]
When compared to the mean relaxation time of 100 sec, r~in is small but not negligible. The unique behavior of the +0.8 PEC material at high concentration requires additional discussion. First, the percentage of fast exchanging molecules (k-~ - 300 sec) is relatively high. It is pertinent here to refer to the discussion in Part I (4), where we noted that the high P values observed for this surface at high concentration are in fact greater than would be expected for a close-packed end-on monolayer. If the additional adsorption represents a "second" layer, then molecules in this second layer would have relatively small adsorption energies. Second, the slow exchange process in this system is considerably slower than for the other experimental conditions (400 min relaxation time compared to 100 to 200 min). A tentative explanation is that this second (slowly exchanging) population of molecules, in order to desorb, has to cross the barrier of the rapidly exchanging surface population which is numerically greater than for the other systems. This requirement would give rise to two markedly different relaxation times as is Journal of Colloid and Interface Science. Vol. 95, No. 1, September 1983
evident in Fig. 3. It would also provide a possible explanation for the different characteristics of the "slow" exchanging populations on the +0.8 PEC at 0.5 mg m1-1 versus the other conditions. Other authors have described behavior which they interpreted as showing the existence of several "populations" of adsorbed molecules (10-12). The evidence in support of such interpretations is usually the observation of partial desorption in response to various treatments of the adsorbed protein. For example, Beissinger and Leonard (10) found that 3,-globulin adsorbed on quartz could be partially desorbed by rinsing with buffer. Dillman and Miller (11) concluded that adsorption of various proteins on neutral and negatively charged surfaces takes place in two distinct ways, distinguished by whether or not the protein is removed by 0.1 N NaOH. Very recently, Robertson et al. (12) showed via exchange experiments using fluorescently labeled proteins that for both albumin and fibrinogen adsorbed on silicone rubber, there are reversibly and irreversibly adsorbed populations. In the discussion thus far, we have not considered the two key parameters characterizing the different interfaces, namely, their net electrical fixed charge and their hydrophobicity (related to swelling in water). Apparently, as was found for the adsorption isotherms (4) none of these parameters has a strong influence on self exchange. If we consider the total percentage exchangeable fibrinogen (denoted pX = p~ + P2), w e see that at low concentration, it is the -0.8 PEC which displays the highest pX value, about twice that for the other surfaces. Since this surface is the most hydrophilic and its net electrical charge is of the same sign as the fibrinogen molecule at pH 7.35, it may be that detachment of an adsorbed molecule is relatively easy. At high concentration, this argument apparently does not hold and we observe the sequence: pZ+o.8> pT-o8 > p~e,~
INTERACTIONS OF FIBRINOGEN WITH POLYELECTROLYTECOMPLEXES
35
to be compared with the sequence of F values in the same concentration regime (4):
so that the rate constant k associated with this model is:
/~+0.8 > ~-0.8 ~> Fneut.
k = ]~eCB .
If the present results are compared with those of Brash and Samak (2), the main conclusion is that exchange is fast in the present systems compared to the polyethylene/albumin system studied by these authors. The turnover of fibrinogen adsorbed on glass surfaces (13) is more akin to the present systems in terms of quantities and rates. This is not surprising since as already noted some similarity exists in the adsorption behavior. Finally, if we consider the mechanism of the protein turnover, the present work leads essentially to the same conclusions as those of Brash and Samak. Two schemes can be postulated to describe exchange between surface and solution. The related chemical equations are as follows:
However, because of the poor reproducibility of data from one experiment to another, it was not possible precisely to check the validity of Eq. [ 12] in the high concentration (isotherm plateau) regime, where CA + CA* (and r = (CA + CA*) A, A being the thickness of the layer) remains almost constant with change in cB. One may conclude from the present set of experiments that for bulky molecules, the exchange between interface and solution is probably more complex than implied by Eq. [9]. Two observations point clearly to this conclusion: (i) we often had to consider the existence of three adsorbed populations in the data analysis and (ii) the rate of the slow exchange process does not depend on bulk concentration, with one exception where the variation is opposite to what Eq. [12] predicts. More experimental data are obviously required if the mechanisms are to be described in detail.
B+A* ~B*+A
[9]
L and
/~ A* ~ B* A ~ B
[10]
where A and B refer to adsorbed and bulk molecules, respectively, with the asterisk denoting radiolabeled molecules. Equations [10] describe an isomerization mechanism, that is, an exchange across a potential barrier. It should be noted that via the structure of the adsorbed layer, rate constants /q could depend indirectly on the concentration of the bathing solution, although proportionality is not expected. Therefore, the fact that no desorption is observed in the presence of pure buffer solution, a result quite general with hydrophobic surfaces (2, 3, 14, 15) strongly indicates that this model is not appropriate to describe exchange. If one works under quasi-open system conditions as in the present work, with cB, - 0, the chemical flux related to Eq. [9] is given by the equation:
dCA, = /~eCBCA* dt
[ 11 ]
[12]
SUMMARY In the present work, dynamic aspects of the interaction between fibrinogen and model polyelectrolyte-complex (PEC) surfaces was investigated, the static aspect having been studied previously (4). The method used was similar to that developed previously by Brash and co-workers (1, 2). It consists of carrying out preliminary adsorption with radiolabeled proteins and subsequently exposing the surface to a solution of nonlabeled molecules. The radioactivity of the surface is followed continuously as a function of time and, in this way, a precise time-dependence of the related processes can be obtained. This technique enabled us to show that in the pseudo-plateau region of the isotherm, exchange between solution and interface takes place at two significantly different rates, so that three populations of adsorbed fibrinogen have to be considered: nonexchanging, rapidly exchanging (small percentage, except Journal of Colloid and Interface Science,
Vol. 95, No. 1, Sep~ernber / 983
36
BRASH ET AL.
for the positively charged PEC surface), and slowly exchanging. In the lower concentration region, the neutral and negative PECs show only one, relatively slow, exchange process (two surface populations) whereas the positive PEC again shows a fast and a slow exchange process. Unique behavior of the positive PEC was also observed with regard to its adsorption isotherm (4). Thus, taking the present results together with those published previously (1, 2) it is possible to claim as a general rule that the "equilibrium" between an adsorbed protein layer and its solution is only partly reversible, since we find that part of the surface layer is exchangeable and part is not. It is worth noting that this conclusion emerges from experiments conducted over much different time intervals: hours (this work with fibrinogen) versus days (Brash et al., with albumin). Some interesting comparisons can be made with previous studies of the same systems (4). In particular, the positively charged PEC interface displays unique behavior from both static and dynamic points of view. To explain the high values of interfacial concentration on this surface, we proposed the existence of a second layer consisting of loosely attached molecules. This seems to be confirmed by the exchange results, where the percentage of fast exchanging proteins is considerably greater than for other surfaces. Finally, it should be mentioned that no obvious correlation could be found between exchange behavior and the varying characteristics of the different PEC surfaces, namely, charge and hydrophobicity.
Journal of Colloid and Interface Science. Vol. 95, NO. 1, September 1983
ACKNOWLEDGMENTS The authors are grateful for financial support of this work by the Medical Research Council of Canada, the Ontario Heart Foundation, and the Centre National de la Recherche Scientifique (France).
REFERENCES 1. Brash, J. L., Uniyal, S., and Samak, Q., Trans. Amer. Soc. Artif Int. Organs 20, 69 (1974). 2. Brash, J. L., and Samak, Q. M., J. Colloid Interface Sci. 65, 495 (1978). 3. Norde, W., in "Adhesion and Adsorption of Polymers" (L. H. Lee, Ed.), p. 801. Plenum, New York, 1980. 4. Schmitt, A., Varoqui, R., Uniyal, S., Brash, J. L., and Pusineri, C., J. Colloid Interface Sci. 92, 25 (1983). 5. Vroman, L., Adams, A. L., Fischer, G. C., and Munoz, P. C., Blood55, 156 (1980). 6. Basse-Cathalinat, B., Baquey, C., Llabrador, Y., and Fleury, A., Int. J. Appl. Rad. Isot. 31, 747 (1980). 7. Ihlenfeld, J. V., and Cooper, S. L., J. Biomed. Mater. Res. 13, 577 (1979). 8. Morrissey, B. W., and Stromberg, R. R., J. Colloid Interface Sci. 46, 152 (1974). 9. Grant, W. H., Morrissey, B. W., and Stromberg, R. R., in "Adhesion Science and Technology" (L. H. Lee, Ed.), Vol. 9A, pp. 43-54. Plenum Press, New York, 1975. 10. Beissinger, R. L., and Leonard, E. F., ASA[O J. 3, 160 (1980). 11. Dillman, W. J., and Miller, I. F., J. Colloidlnterface Sci. 44, 221 (1973). 12. Lok, B. K., Cheng, Y. L., and Robertson, C. R., J. Colloid Interface Sci. 91, 104 (1983). 13. Chan, B. M. C., and Brash, J. L., J. Colloid Interface Sci. 82, 217 (1981). 14. Brash, J. L., and Lyman, D. J., J. Biomed. Mater. Res. 3, 175 (1969). 15. Morrissey, B. W., Ann. iV. Y. Acad. ScL 283, 50 (1977).