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The adsorption of human plasma albumin on solid surfaces, with special attention to the kinetic aspects
The Adsorption of Human Plasma Albumin on Solid Surfaces, with Special Attention to the Kinetic Aspects P E G G Y V A N D U L M AND W I L L E M N O R D E 1 Laboratory for Physical and Colloid Chemistry, Agricultural University, De Dreijen 6, 6703 BC Wageningen, The Netherlands
Received January 4, 1982; accepted April 16, 1982 The adsorption of human plasma albumin, labeled with iodine-125, was studied under various conditions of sorbent hydrophobicity and charge on the sorbent and the protein. The adsorption on a hydrophobic, siliconized, glass surface was shown to be higher than on a hydrophilic glass surface. The influence of charge-charge interactions was investigated by performing the experiments at pH 4.0 (positivelycharged albumin) and at pH 7.4 (negativelycharged albumin) and by using positively and negatively charged polystyrene surfaces. The rate of adsorption seems to be controlled by the diffusion of the protein from the solution toward the sorbent surface, unless both the albumin and the surface are negatively charged. Under those conditions the albumin molecules have to overcome an energy barrier before they are deposited. In some cases fast initial adsorption is followed by desorption of a fraction of the albumin molecules. This phenomenon seems to be related to the relative rates of deposition and of structural rearrangements in the adsorbed protein molecules. INTRODUCTION
ecule, in such a way that it attaches an inDue to their amphiphilic character pro- creasing n u m b e r of segments per unit area of sorbent surface. This might explain the teins are highly surface-active. Over a wide low desorbability of proteins usually obrange of conditions they adsorb at almost any served, even where the adsorption isotherm interface. The overall adsorption process does not show high-affinity character. m a y be considered as the c o m p o u n d e d result Proteins adsorbing at solid surfaces m a y of various intricate subprocesses (1-5). U n as well change their structure (8, 9). With der suitable conditions the contribution from solid surfaces, however, this cannot be m o n the various constituting factors m a y be established from a t h e r m o d y n a m i c analysis (6). itored by following the change of surface In this way the effects of physicochemical pressure with time. Alterations in protein structure resulting from adsorption m a y be properties of both the protein and the sorbent detected using spectroscopic methods. In surface on their mutual affinity can be traced. particular fluorescence spectroscopy (10, 1 1), Such a t h e r m o d y n a m i c analysis, however, infrared spectroscopy (12), and ESCA (13) does not lead to conclusions with regard to seem the most promising techniques. H o w the kinetics of the sorption process. In the ever, most of the information concerning case of liquid interfaces it has clearly been rearrangements in the protein structure is indemonstrated that, while the a m o u n t adferred indirectly from adsorption, desorpsorbed reaches a constant value relatively quickly, the surface pressure approaches a tion, and exchange studies (8, 14-19). In this article we will discuss the influence steady-state value only after m u c h longer o f electric charge and hydrophobicity o f the times (e.g. (7)). This points to slow structural sorbent surface on the adsorption of h u m a n rearrangements in the adsorbed protein molplasma albumin that is tagged with an iodine To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . label. 248 0021-9797/83/010248-08503.00/0 Copyright © 1983 by Academic Press, Inc. All fights of reproduction in any form reserved.
Journal of Colloid and Interface Science, VoL 91, No. 1, January 1983
ADSORPTION OF ALBUMIN MATERIALS AND METHODS Human plasma albumin labeled with iodine-125 (125I-labeled HPA) was obtained from Amersham, supplied in an aqueous solution containing 20 mg protein/cm 3. Its specific activity directly after labeling amounts to 2.5 uCi/mg of albumin. Nonradioactive HPA was purchased from Sigma Chemical Co. (Fraction V). Prior to use it was dialyzed against water for 2 days at 4°C. All other chemicals were of analytical grade and the water was deionized and distilled from an allPyrex apparatus. Glass slides (24 × 50 × 0.15 mm 3) were obtained from Chance Propper Ltd., Smethwick, Warley, England. They were cleaned according to the method described in (19). Hydrophobic surfaces were prepared by siliconizing glass slides, according to the procedure described in Ref. (19). The slides were stored in methanol. Polystyrene slides were prepared in the following way. Polystyrene latices were produced according to the procedures described in (20) and (21). The surface charge density at the negatively charged latex (-OSO3 groups) amounts to -15/~C cm -a and at the positively charged latex (-N-C(CH3)2C(NH2)~- groups) to ca. +4 uC cm -2. The latices were dialyzed against water for 2 weeks, freeze-dried, and the polystyrene was dissolved in toluene to a concentration of 7% (w/w). One hundred cubic centimeters of this solution was poured into a glass dish (q~ 22.3 cm) with a fiat bottom, that is mounted exactly horizontally. Air bubbles were removed and the toluene was allowed to evaporate slowly over a period of 3 days. In this way polystyrene films are formed that contain either positively or negatively charged groups. Obviously, as a result of reorientations of these groups during film formation, the charge density of the film is likely to be different from that of the latex that is used as the source. The obtained polystyrene film was cut into slides (45.0 × 21.7 mm 2) that were stored dust-free.
249
The adsorption experiments were performed in a manner similar to those described by Penners et al. (19). In our design the perspex block contained 15 compartments, each having dimensions of 60 × 45 × 10 mm 3. The test slides were incubated in the compartments with a standard incubation volume of l 8 ml at room temperature. Experiments were performed at two different pH values, i.e., pH 4.0 where the albumin is positively charged, and pH 7.4 where its charge is negative. The pH values were adjusted with acetic acid buffer and Tris buffer, respectively. In those cases where the total ionic strength exceeded 0.01 M, NaC1 was added. During incubation the block was tilted from one side to the other so that the slides changed their positions with each tilt and the solution was in continuous motion. After incubation for the desired period of time the slides were "washed" by dipping them four subsequent times in 250 cm 3 of the corresponding fresh buffer solution. In this way the protein solution adhering to the slides, after taking them from the incubation medium, was removed. No evidence was obtained that on passing the slides through the air-water interface albumin molecules adsorbed on the slides were transferred to the air-water interface. The samples were airdried, transferred to adhesive tape, and then curled into a roll that was brought into a y-counting tube. The 3,-emission was counted in a one-channel Philips PW 4003 spectrometer equipped with a NaI (T l) well-type crystal of 2 × 1.75 in. 2. To determine the efficiency, known amounts of ~25I-labeled HPA were brought on the slides that were then treated identically to the slides sampled from the incubation solution (except for the washing procedure). RESULTS
AND
DISCUSSION
Adsorption as a Function of Supplied Protein Concentration
Adsorption isotherms have been determined for the untreated and siliconized glass Journal of Colloid and Interface Science, Vol. 91, No. 1, January 1983
250
VAN DULM AND NORDE X
2-rag
m -2
x
x
X
X ~
0 0
1
/¢~ /
0
°''~'-~ I
I
0.1
0.2
0
l
CHPA
0.3 g drn-3
FIG. 1. Adsorption of '251-labeled HPA on glass (O) and siliconized glass (×) surfaces. T = 22°C; pH 7.4; ionic strength 0.05 M.
surfaces, at pH 7.4 and 0.05 Mionic strength. The incubation time was ca. 18 hr. Various authors have reported on the effect of an iodine label on the adsorption behavior of proteins. Their results are rather controversial. For instance, Van der Scheer (22) finds a preferred adsorption of labeled albumin molecules over the nonlabeled ones. He states that the preference strongly depends on the labeling procedure. This suggests that it is not the mere presence of the iodine atom but rather the changes in the albumin structure resulting from the labeling procedure that cause the effect on adsorption. Other authors (16, 17) have not observed an effect of iodination on the adsorption characteristics of plasma albumin and other proteins. We have checked the influence of the ~25I label by comparing the radioactivities in layers of albumin adsorbed from solutions containing a radioactive albumin fraction of 80 and 1%, respectively. This has been done at two initial albumin concentrations, i.e., 0.25 and 1.50 g dm -3, and using glass and siliconized glass as the sorbents. For each albumin dosage the radioactivities at the sorbent surface were proportional to those in solution. Assuming that the net amount of albumin adsorbed is not affected by the fraction of labeled molecules in solution (both albumin concentrations lead to plateau adsorption), the results indicate that neither the labeled nor the nonlabeled albumin molecules are preferentially adsorbed. Journal of Colloid andlnterface Science, Vol. 91, No. 1, January 1983
The isotherms obtained are shown in Fig. 1. The plateau value is higher for the more hydrophobic surface. This trend has also been reported by other authors (e.g., 19, 23, 24). The affinity between the protein and the sorbent as judged from the initial part of the isotherm is higher for the more hydrophobic surface. This difference probably must be attributed to the different contributions from sorbent dehydration to the overall adsorption.
Desorption Glass slides (both untreated and siliconized) were exposed for 18 hr to a 0.3 g dm -3 albumin solution ofpH 7.4 and 0.05 M ionic strength. The protein-saturated slides were then transferred to a buffer solution of pH 7.4 and 0.05 M. In neither case could desorption of albumin be detected over a period of 2 days. The observation, as.with the hydrophilic untreated glass surface, of a relatively low-affinity adsorption isotherm and a high-affinity desorption isotherm, is indicative of structural changes in the adsorbed protein molecule, so that its affinity for the surface increases with time. If both the adsorption and desorption isotherm are of the high-affinity type, as observed for the siliconized glass surface, the results are not conclusive as to the occurrence of such conformational changes.
AdsorptionKinetics Rates of adsorption have been determined under various conditions with respect to hydrophobicity and electrical charge of the sorbent surface, electrical charge on the protein molecule, and ionic strength of the medium. In each experiment the supplied HPA concentration was 38 mg dm -3. The results are shown in Figs. 2, 3, and 4. It can be seen that the adsorption proceeds rather quickly. The time to attain steady-state adsorption depends on the conditions, but in none of the experiments does it exceed 1 hr. Since the sorbent surface, when first ex-
251
ADSORPTION OF A L B U M I N
2 - r a g m -2 V
Lx...~-' x X
~x
~r .o~. o......-o- - - ' - - - °
O"
O - -
oO.o~° d
incubation t i m e ( t )
I
4O
80
120 min
FaG. 2. Rates of adsorption of 1251-labeled HPA on glass (©) and siliconized glass (×) surfaces. Concentration of 125I-labeled HPA 39 mg dm-3; T = 22°C; p H 7.4; ionic strength 0.05 M.
In Figs. 5, 6, and 7 F is plotted vs. t ~/2 for the various sorbent surfaces. Since in each experiment the first sample was taken after 0.5 min, the initial part of the curves has to be established by interpolation between 0 and 0.71 rain ~/2. In the figures these parts of the curves are dashed. The values for D derived from the initial slopes are, therefore, subject to a large uncertainty. In spite of that it can be concluded that only with the positively charged polystyrene surface and perhaps with the negatively charged polystyrene surface at p H 4 the initial adsorption rates are in agreement with a diffusion-controlled rate based on the diffusion constant quoted in the literature (D = 7.0 × 10 -~1 m 2 sec -~
posed to the protein solution, is void of adsorbed protein molecules, the initial rate o f adsorption is: 8F = 2c(D/Tr)mOt 1/2
[ 1]
where c is the protein concentration in solution, D the diffusion coefficient, t the incubation time, and 7r is 3.14. This equation assumes a diffusion-controlled process. Although in our experiments the solutions were gently mixed, there will be an undisturbed layer adjacent to the sorbent surface, where mass transport occurs only by diffusion. Thus, for a given protein concentration the initial slope o f the plot o f 1~ vs. t 1/2 determined by the diffusion coefficient D.
mg m -z
1' b
x x x ~"'x
/
x
,,x
,
x
x
,,f
I 40
I 80
i incubation time(t) 120 rain
FIG. 3. Rates o f adsorption o f =25I-labeled H P A on negatively charged polystyrene surfaces. Concentration o f J25I-labeled HPA 38 mg din-3; T = 22°C; ionic strength 0.01 M; p H 7.4 (×) and p H 4.0
(o). Journal of Colloid and Interface Science,
Vol. 91, No. 1, January 1983
252
VAN DULM AND NORDE m g m -2
0
o
I
I
4o
80
,&, t
• .b
0
lincubation time(t) 120 m i n
FIG. 4. Rates of adsorption of t25I-labeled HPA on positively charged polystyrene surfaces. Concentration of 12SI-labeledHPA 38 mg dm-3; T = 22°C; ionic strength 0.001 M, pH 7.4 (O); ionic strength 0.001 M, pH 4.0 (©); ionic strength 0.1 M , pH 7.4 (A); ionic strength 0.1 M, pH 4.0 (A). (25)). I n t h e o t h e r cases, i.e., t h e u n t r e a t e d a n d the siliconized glass surfaces at p H 7.4 a n d the n e g a t i v e l y c h a r g e d p o l y s t y r e n e surface at p H 7.4, t h e a d s o r p t i o n is m u c h slower. T h i s i m p l i e s t h a t o n l y a f r a c t i o n o f t h e alb u m i n m o l e c u l e s t h a t r e a c h t h e s o r b e n t surface b y diffusion is a c t u a l l y d e p o s i t e d at t h a t surface. It c o u l d b e t h a t o n l y t h o s e m o l e c u l e s t h a t arrive at t h e s o r b e n t surface in a specially suitable o r i e n t a t i o n c a n u n d e r g o a d s o r p t i o n . A n o t h e r possibility is t h a t t h e a l b u m i n m o l ecules h a v e to cross s o m e b a r r i e r b e f o r e t h e y
a t t a c h at t h e s o r b e n t surface. T h e n a t u r e o f the b a r r i e r can, in p r i n c i p l e , be q u i t e different. F o r instance, it m a y b e t h e result o f electrostatic r e p u l s i o n , b u t also o f steric r e p u l sion. T h e s y s t e m s for w h i c h we observe relatively slow a d s o r p t i o n h a v e in c o m m o n t h a t b o t h t h e s o r b e n t surface a n d t h e p r o t e i n
m g m -2
•
•
2 - m g m -2
x r"
x 1
x
x
x
I ~.I
x
o..~°-"~ ~
1
2
I 3
,~
rain1'2
FIG. 5. Adsorption of 125I-labeledHPA as a function of t t/2 on glass (©) and siliconized glass (×) surfaces. Conditions as in Fig. 2. Initial slope calculated for a diffusion coefficient of 7.0 × 10-~ m 2 sec-~ is represented by the straight line. Journal of Colloid and Interface Science, Vol. 91, No. 1, J a n u a r y 1983
0
1
2
3
4 r a i n +lz
FIG. 6. Adsorption of 125I-labeledHPA as a function of t v2 on negatively charged polystyrene surfaces. Conditions and symbols as in Fig. 3. The straight line represents the initial slope calculated for a diffusion coefficient of 7.0 X 10-H m 2 sec-1.
ADSORPTION OF ALBUMIN
253
mg m -2
served. The reason for it may be, first, that the electrokinetic charge on the HPA molecule at pH 4 is much smaller than at pH 7.4 (3) and, second, that the albumin molecule tends to adsorb with its negative groups oriented toward the sorbent surface. The latter reason may also explain the finding that the / initial rate of adsorption of positively charged / albumin (pH 4) is slower on a negatively charged polystyrene surface than on a positive one. Since at the various surfaces, at otherwise similar conditions, the albumin molecule may undergo structural rearrangements to different extents, the steady-state values of P cannot be simply interpreted as a measure I I I I for the affinity between the protein and the 0 1 2 3 4 rain ~lz sorbent. For both the positive and the negFIG. 7. Adsorption of 125I-labeled H P A as a function ative polystyrene surface P is higher at pH of t ~/2 on positively charged polystyrene surfaces. Con4 than at pH 7.4. This is in agreement with ditions and symbols as in Fig. 4. The straight line represents the initial slope calculated for a diffusion coefalbumin adsorption at negatively charged ficient of 7.0 × 10 l~ m 2 see-L particles of, e.g., polystyrene (1), silver iodide (26), glass (27), and at the positively charged molecule are negatively charged. We are particles of hematite (28). The variation of therefore inclined to conclude that the slow F with pH has been interpreted in terms of adsorption is due to an energy barrier caused structural rearrangements in the albumin by overlapping electric fields from the neg- molecule (1-5). Although the adsorptions at ative charges on the sorbent and the protein. the positively charged polystyrene slides fit In that case, the rate of adsorption is slowed in this picture, the influence o f p H is different down by a factor e - A / k T where A is the ac- from that observed with positive polystyrene tivation energy required to overcome the bar- latices (29). With the latex the albumin adtier and k T is one unit of thermal energy. sorption at pH 7.4 is significantly larger than Hence, A can be evaluated by comparing the at pH 4. It should be realized, however, that initial slope of the curve for P (t 1/2) with the as a result of the preparation of the slides one obtained for a diffusion-controlled pro- from the latex the surface charge density may cess with D = 7.0 × 10TM m 2 sec -l. The val- be affected considerably. The influence of ionic strength has been ues thus derived for A are 2.9 k T and 0.8 k T for adsorption on untreated and siliconized studied for the adsorption on positive polyglass, respectively, both at pH 7.4 and 0.05 styrene only. At pH 4 as well as pH 7.4 adM ionic strength. For the adsorption at the sorptions are larger at higher ionic strength. negatively charged polystyrene surface at Referring to our previous studies with the 0.01 M ionic strength, the values for A are albumin-polystyrene latex systems, this ionic strength effect may be due to different degrees 1.6 k T at pH 7.4 and 0.4 k T at pH 4.0. In analogy to the above reasoning, an elec- of protein reconformation rather than to diftric energy barrier would be expected for the ferences in lateral repulsion between the adadsorption of positively charged albumin sorbed albumin molecules (1-5). The finding (pH 4) on a positively charged polystyrene that the influence of the ionic strength is surface. This, however, has not been ob- more pronounced at pH 4 than at pH 7.4,
f:
Journal of Colloid and Interface Science, Vol. 91, No. i, January 1983
254
VAN DULM AND NORDE
whereas the protein charge at pH 7.4 is larger, supports that conclusion. There is one more remarkable feature to be discussed. In some cases, namely at pH 4 and not too low ionic strength, the F(t) curves pass through a maximum at about 20 to 30 min. A few times a similar behavior has been reported in the literature. For instance, Walton and Soderquist (8) find such a maximum for the adsorption of bovine serum albumin on siliconized glass and on glass coated by a copolymer of lysine and leucine. Furthermore, Horbett (16), studying the competitive adsorption between proteins from blood plasma onto different surfaces, reports a significant desorption of albumin from a hydrophobic surface following a fast initial adsorption. He assumes that after prolonged incubation albumin is exchanged against other plasma proteins. In the experiments described in this paper as well as in Ref. (8), albumin is the only protein present, and it can, therefore, not be displaced by a different kind of protein. However, the albumin sample itself is not completely homogeneous, i.e., it may contain a small fraction of dimers and higher aggregates. Furthermore, the number of 0.7 sulfhydrylgroups per albumin molecule (30) points to the coexistence of mercaptalbumin and nonmercaptalbumin. Hence, there are a number of different species present that compete for adsorption. This could explain the adsorption maximum with time, although in that case the influence o f p H and ionic strength on the occurrence of that maximum is still to be explained. Another possibility is that desorption after some time is the result of rearrangements in the structure of already adsorbed albumin molecules. As has been mentioned in the introduction, adsorbed protein molecules may change their conformation in order to attach more segments at the sorbent surface, i.e., to optimize their surface bonding. Since the individual segments are reversibly adsorbed some protein molecules may become completely detached in favor of the "spreading" of other adsorbed moleJournal of Colloid and Interface Science, VoI.91, No. 1, January 1983
cules. Whether or not such structural rearrangements lead to some desorption depends on the relative rates of deposition of the protein on the sorbent surface and of the structural rearrangements, respectively. Thus, a maximum in P(t) is more likely to show up in cases of fast deposition and slow reconformation. Indeed, as is shown in Figs. 2-4, at pH 4 the adsorption proceeds faster than at pH 7.4. Moreover, as a result of the lower charge density, the internal coherence in the albumin molecule at pH 4 is expected to be stronger than at pH 7.4, which would imply a greater resistance to conformational changes. The absence of a peak in the F(t) curve at pH 4 and 10-3 M could then be ascribed to a reduced screening of interactions between charges at that low ionic strength. CONCLUSION
Albumin adsorption is markedly influenced by the properties of the sorbent surface. The amount of protein accommodated in the adsorbed layer is larger at a more hydrophobic surface. The adsorbed amount is furthermore determined by the ratio between the rates of deposition and the rate of structural rearrangements in the adsorbed molecule. A high value for this ratio results in higher adsorptions. The kinetics of the adsorption process depend strongly on the electric interactions. More specifically, if both the protein and the sorbent are negatively charged, the initial rate of adsorption is relatively low, whereas at other conditions it corresponds to that predicted from diffusion theory. In some cases, fast initial adsorption is followed by some desorption. This desorption of a fraction of the protein may be a consequence of progressive unfolding of the remaining adsorbed albumin molecules. ACKNOWLEDGMENT The authors gratefully acknowledge the cooperation of the Institute for the Application of Atomic Science in Agriculture, Wageningen, The Netherlands, which granted the facilities to perform the experiments involving radionuclides.
ADSORPTION OF ALBUMIN REFERENCES 1. Norde, W., and Lyklema, J., J. ColloidlnterfaceSci. 66, 257 (1978). 2. Norde, W., and Lyklema, J., J. ColloidlnterfaceSci. 66, 266 (1978). 3. Norde, W., and Lyklema, J., J. ColloidlnterfaceSci. 66, 277 (1978). 4. Norde, W., and Lyklema, J., J. Colloidlnterface Sei. 66, 286 (1978). 5. Norde, W., and Lyklema, J., Jr. ColloidlnterfaceSci. 66, 295 (1978). 6. Norde, W., and Lyklema, J., J. ColloidlnterfaeeSci. 71, 350 (1979). 7. Adams, D. J., Evans, M. T. A., Mitchell, J. R., Phillips, M. C., and Rees, P. M., J. Polym. Sci. C34, 167 (1971). 8. Walton, A. G., and Soderquist, M. E., Croatica Chem. Acta 53, 363 (1980). 9. Chan, B. M. C., and Brash, J. L., J. Colloidlnterface Sci. 84, 263 (1981). 10. Watkins, R. W., and Robertson, C. R., J. Biomed. Mater. Res. 11, 915 (1977). 11. Walton, A. G., and Maenpa, F. C., J. Colloid Interface Sci. 72, 265 (1979). 12. Gendreau, R. M., Appl. Spectrosc. 35, 353 (1981). 13. Ratner, B. D., Horbett, T. A., Shuttleworth, D., and Thomas, H. R., J. Colloid Interface Sci. 83, 630 (1981). 14. Grant, W. H., and Dehl, R. E., in "Adhesion and Adsorption of Polymers" (Lieng-Huang Lee, Ed.), Part B, p. 827. Plenum Press, New York, 1980.
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15. Brynda, E., Houska, M., Pokornh, Z., Cepalova, N. A., Moiseev, Yu. V., and Kalal, J., J. Bioeng. 2, 411 (1978). 16. Horbett, T. A., in "Adhesion and Adsorption of Polymers" (Lieng-Huang Lee, Ed.), Part B, p. 677. Plenum Press, New York, 1980. 17. Brash, J. L., and Samak, Q., J. ColloidlnterfaceSci. 65, 495 (1978). 18. Chan, B. M. C., and Brash, J. L., J. Colloidlnterface Sci. 82, 217 (1981). 19. Penners, G., Priel, Z., and Silberberg, A., 31 Colloid Interface Sci. 80, 437 (1981). 20. Furusawa, K., Norde, W., and Lyklema, J., KolloidZ. Z. Polym. 250, 908 (1972). 21. Goodwin, J. W., Ottewill, R. H., and Pelton, R., Colloid Polym. Sci. 257, 61 (1979). 22. Van der Scheer, A., Thesis, Technical University Twente, The Netherlands, 1978. 23. MacRitchie, F., ,L Colloid Interface Sci. 38, 484 (1972). 24. Brash, J. L., and Davidson, V. J., Thrornb. Res. 9, 249 (1976). 25. Keller, K. H., Canales, E. R., and Yum, S. I., J. Phys. Chem. 75, 379 (1971). 26. Matuszewska, B., Norde, W., and Lyklema, J., Z Colloid Interface Sci. 84, 403 (1981). 27. Hull, H. B., Biochim. Biophys. Acta 19, 464 (1956). 28. Koutsoukos, P., Lyklema, J., and Norde, W., J. Colloid Interface Sci., submitted. 29. Koutsoukos, P., Mumme-Young, C. A., Norde, W., and Lyklema, J., Colloids and Surfaces, 5, 93 (1982). 30. Andersson, L., Biochim. Biophys. Acta 117, 115 (1966).
Journal of Colloid and Interface Science, Vol. 91, No. 1, January 1983
Received January 4, 1982; accepted April 16, 1982 The adsorption of human plasma albumin, labeled with iodine-125, was studied under various conditions of sorbent hydrophobicity and charge on the sorbent and the protein. The adsorption on a hydrophobic, siliconized, glass surface was shown to be higher than on a hydrophilic glass surface. The influence of charge-charge interactions was investigated by performing the experiments at pH 4.0 (positivelycharged albumin) and at pH 7.4 (negativelycharged albumin) and by using positively and negatively charged polystyrene surfaces. The rate of adsorption seems to be controlled by the diffusion of the protein from the solution toward the sorbent surface, unless both the albumin and the surface are negatively charged. Under those conditions the albumin molecules have to overcome an energy barrier before they are deposited. In some cases fast initial adsorption is followed by desorption of a fraction of the albumin molecules. This phenomenon seems to be related to the relative rates of deposition and of structural rearrangements in the adsorbed protein molecules. INTRODUCTION
ecule, in such a way that it attaches an inDue to their amphiphilic character pro- creasing n u m b e r of segments per unit area of sorbent surface. This might explain the teins are highly surface-active. Over a wide low desorbability of proteins usually obrange of conditions they adsorb at almost any served, even where the adsorption isotherm interface. The overall adsorption process does not show high-affinity character. m a y be considered as the c o m p o u n d e d result Proteins adsorbing at solid surfaces m a y of various intricate subprocesses (1-5). U n as well change their structure (8, 9). With der suitable conditions the contribution from solid surfaces, however, this cannot be m o n the various constituting factors m a y be established from a t h e r m o d y n a m i c analysis (6). itored by following the change of surface In this way the effects of physicochemical pressure with time. Alterations in protein structure resulting from adsorption m a y be properties of both the protein and the sorbent detected using spectroscopic methods. In surface on their mutual affinity can be traced. particular fluorescence spectroscopy (10, 1 1), Such a t h e r m o d y n a m i c analysis, however, infrared spectroscopy (12), and ESCA (13) does not lead to conclusions with regard to seem the most promising techniques. H o w the kinetics of the sorption process. In the ever, most of the information concerning case of liquid interfaces it has clearly been rearrangements in the protein structure is indemonstrated that, while the a m o u n t adferred indirectly from adsorption, desorpsorbed reaches a constant value relatively quickly, the surface pressure approaches a tion, and exchange studies (8, 14-19). In this article we will discuss the influence steady-state value only after m u c h longer o f electric charge and hydrophobicity o f the times (e.g. (7)). This points to slow structural sorbent surface on the adsorption of h u m a n rearrangements in the adsorbed protein molplasma albumin that is tagged with an iodine To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . label. 248 0021-9797/83/010248-08503.00/0 Copyright © 1983 by Academic Press, Inc. All fights of reproduction in any form reserved.
Journal of Colloid and Interface Science, VoL 91, No. 1, January 1983
ADSORPTION OF ALBUMIN MATERIALS AND METHODS Human plasma albumin labeled with iodine-125 (125I-labeled HPA) was obtained from Amersham, supplied in an aqueous solution containing 20 mg protein/cm 3. Its specific activity directly after labeling amounts to 2.5 uCi/mg of albumin. Nonradioactive HPA was purchased from Sigma Chemical Co. (Fraction V). Prior to use it was dialyzed against water for 2 days at 4°C. All other chemicals were of analytical grade and the water was deionized and distilled from an allPyrex apparatus. Glass slides (24 × 50 × 0.15 mm 3) were obtained from Chance Propper Ltd., Smethwick, Warley, England. They were cleaned according to the method described in (19). Hydrophobic surfaces were prepared by siliconizing glass slides, according to the procedure described in Ref. (19). The slides were stored in methanol. Polystyrene slides were prepared in the following way. Polystyrene latices were produced according to the procedures described in (20) and (21). The surface charge density at the negatively charged latex (-OSO3 groups) amounts to -15/~C cm -a and at the positively charged latex (-N-C(CH3)2C(NH2)~- groups) to ca. +4 uC cm -2. The latices were dialyzed against water for 2 weeks, freeze-dried, and the polystyrene was dissolved in toluene to a concentration of 7% (w/w). One hundred cubic centimeters of this solution was poured into a glass dish (q~ 22.3 cm) with a fiat bottom, that is mounted exactly horizontally. Air bubbles were removed and the toluene was allowed to evaporate slowly over a period of 3 days. In this way polystyrene films are formed that contain either positively or negatively charged groups. Obviously, as a result of reorientations of these groups during film formation, the charge density of the film is likely to be different from that of the latex that is used as the source. The obtained polystyrene film was cut into slides (45.0 × 21.7 mm 2) that were stored dust-free.
249
The adsorption experiments were performed in a manner similar to those described by Penners et al. (19). In our design the perspex block contained 15 compartments, each having dimensions of 60 × 45 × 10 mm 3. The test slides were incubated in the compartments with a standard incubation volume of l 8 ml at room temperature. Experiments were performed at two different pH values, i.e., pH 4.0 where the albumin is positively charged, and pH 7.4 where its charge is negative. The pH values were adjusted with acetic acid buffer and Tris buffer, respectively. In those cases where the total ionic strength exceeded 0.01 M, NaC1 was added. During incubation the block was tilted from one side to the other so that the slides changed their positions with each tilt and the solution was in continuous motion. After incubation for the desired period of time the slides were "washed" by dipping them four subsequent times in 250 cm 3 of the corresponding fresh buffer solution. In this way the protein solution adhering to the slides, after taking them from the incubation medium, was removed. No evidence was obtained that on passing the slides through the air-water interface albumin molecules adsorbed on the slides were transferred to the air-water interface. The samples were airdried, transferred to adhesive tape, and then curled into a roll that was brought into a y-counting tube. The 3,-emission was counted in a one-channel Philips PW 4003 spectrometer equipped with a NaI (T l) well-type crystal of 2 × 1.75 in. 2. To determine the efficiency, known amounts of ~25I-labeled HPA were brought on the slides that were then treated identically to the slides sampled from the incubation solution (except for the washing procedure). RESULTS
AND
DISCUSSION
Adsorption as a Function of Supplied Protein Concentration
Adsorption isotherms have been determined for the untreated and siliconized glass Journal of Colloid and Interface Science, Vol. 91, No. 1, January 1983
250
VAN DULM AND NORDE X
2-rag
m -2
x
x
X
X ~
0 0
1
/¢~ /
0
°''~'-~ I
I
0.1
0.2
0
l
CHPA
0.3 g drn-3
FIG. 1. Adsorption of '251-labeled HPA on glass (O) and siliconized glass (×) surfaces. T = 22°C; pH 7.4; ionic strength 0.05 M.
surfaces, at pH 7.4 and 0.05 Mionic strength. The incubation time was ca. 18 hr. Various authors have reported on the effect of an iodine label on the adsorption behavior of proteins. Their results are rather controversial. For instance, Van der Scheer (22) finds a preferred adsorption of labeled albumin molecules over the nonlabeled ones. He states that the preference strongly depends on the labeling procedure. This suggests that it is not the mere presence of the iodine atom but rather the changes in the albumin structure resulting from the labeling procedure that cause the effect on adsorption. Other authors (16, 17) have not observed an effect of iodination on the adsorption characteristics of plasma albumin and other proteins. We have checked the influence of the ~25I label by comparing the radioactivities in layers of albumin adsorbed from solutions containing a radioactive albumin fraction of 80 and 1%, respectively. This has been done at two initial albumin concentrations, i.e., 0.25 and 1.50 g dm -3, and using glass and siliconized glass as the sorbents. For each albumin dosage the radioactivities at the sorbent surface were proportional to those in solution. Assuming that the net amount of albumin adsorbed is not affected by the fraction of labeled molecules in solution (both albumin concentrations lead to plateau adsorption), the results indicate that neither the labeled nor the nonlabeled albumin molecules are preferentially adsorbed. Journal of Colloid andlnterface Science, Vol. 91, No. 1, January 1983
The isotherms obtained are shown in Fig. 1. The plateau value is higher for the more hydrophobic surface. This trend has also been reported by other authors (e.g., 19, 23, 24). The affinity between the protein and the sorbent as judged from the initial part of the isotherm is higher for the more hydrophobic surface. This difference probably must be attributed to the different contributions from sorbent dehydration to the overall adsorption.
Desorption Glass slides (both untreated and siliconized) were exposed for 18 hr to a 0.3 g dm -3 albumin solution ofpH 7.4 and 0.05 M ionic strength. The protein-saturated slides were then transferred to a buffer solution of pH 7.4 and 0.05 M. In neither case could desorption of albumin be detected over a period of 2 days. The observation, as.with the hydrophilic untreated glass surface, of a relatively low-affinity adsorption isotherm and a high-affinity desorption isotherm, is indicative of structural changes in the adsorbed protein molecule, so that its affinity for the surface increases with time. If both the adsorption and desorption isotherm are of the high-affinity type, as observed for the siliconized glass surface, the results are not conclusive as to the occurrence of such conformational changes.
AdsorptionKinetics Rates of adsorption have been determined under various conditions with respect to hydrophobicity and electrical charge of the sorbent surface, electrical charge on the protein molecule, and ionic strength of the medium. In each experiment the supplied HPA concentration was 38 mg dm -3. The results are shown in Figs. 2, 3, and 4. It can be seen that the adsorption proceeds rather quickly. The time to attain steady-state adsorption depends on the conditions, but in none of the experiments does it exceed 1 hr. Since the sorbent surface, when first ex-
251
ADSORPTION OF A L B U M I N
2 - r a g m -2 V
Lx...~-' x X
~x
~r .o~. o......-o- - - ' - - - °
O"
O - -
oO.o~° d
incubation t i m e ( t )
I
4O
80
120 min
FaG. 2. Rates of adsorption of 1251-labeled HPA on glass (©) and siliconized glass (×) surfaces. Concentration of 125I-labeled HPA 39 mg dm-3; T = 22°C; p H 7.4; ionic strength 0.05 M.
In Figs. 5, 6, and 7 F is plotted vs. t ~/2 for the various sorbent surfaces. Since in each experiment the first sample was taken after 0.5 min, the initial part of the curves has to be established by interpolation between 0 and 0.71 rain ~/2. In the figures these parts of the curves are dashed. The values for D derived from the initial slopes are, therefore, subject to a large uncertainty. In spite of that it can be concluded that only with the positively charged polystyrene surface and perhaps with the negatively charged polystyrene surface at p H 4 the initial adsorption rates are in agreement with a diffusion-controlled rate based on the diffusion constant quoted in the literature (D = 7.0 × 10 -~1 m 2 sec -~
posed to the protein solution, is void of adsorbed protein molecules, the initial rate o f adsorption is: 8F = 2c(D/Tr)mOt 1/2
[ 1]
where c is the protein concentration in solution, D the diffusion coefficient, t the incubation time, and 7r is 3.14. This equation assumes a diffusion-controlled process. Although in our experiments the solutions were gently mixed, there will be an undisturbed layer adjacent to the sorbent surface, where mass transport occurs only by diffusion. Thus, for a given protein concentration the initial slope o f the plot o f 1~ vs. t 1/2 determined by the diffusion coefficient D.
mg m -z
1' b
x x x ~"'x
/
x
,,x
,
x
x
,,f
I 40
I 80
i incubation time(t) 120 rain
FIG. 3. Rates o f adsorption o f =25I-labeled H P A on negatively charged polystyrene surfaces. Concentration o f J25I-labeled HPA 38 mg din-3; T = 22°C; ionic strength 0.01 M; p H 7.4 (×) and p H 4.0
(o). Journal of Colloid and Interface Science,
Vol. 91, No. 1, January 1983
252
VAN DULM AND NORDE m g m -2
0
o
I
I
4o
80
,&, t
• .b
0
lincubation time(t) 120 m i n
FIG. 4. Rates of adsorption of t25I-labeled HPA on positively charged polystyrene surfaces. Concentration of 12SI-labeledHPA 38 mg dm-3; T = 22°C; ionic strength 0.001 M, pH 7.4 (O); ionic strength 0.001 M, pH 4.0 (©); ionic strength 0.1 M , pH 7.4 (A); ionic strength 0.1 M, pH 4.0 (A). (25)). I n t h e o t h e r cases, i.e., t h e u n t r e a t e d a n d the siliconized glass surfaces at p H 7.4 a n d the n e g a t i v e l y c h a r g e d p o l y s t y r e n e surface at p H 7.4, t h e a d s o r p t i o n is m u c h slower. T h i s i m p l i e s t h a t o n l y a f r a c t i o n o f t h e alb u m i n m o l e c u l e s t h a t r e a c h t h e s o r b e n t surface b y diffusion is a c t u a l l y d e p o s i t e d at t h a t surface. It c o u l d b e t h a t o n l y t h o s e m o l e c u l e s t h a t arrive at t h e s o r b e n t surface in a specially suitable o r i e n t a t i o n c a n u n d e r g o a d s o r p t i o n . A n o t h e r possibility is t h a t t h e a l b u m i n m o l ecules h a v e to cross s o m e b a r r i e r b e f o r e t h e y
a t t a c h at t h e s o r b e n t surface. T h e n a t u r e o f the b a r r i e r can, in p r i n c i p l e , be q u i t e different. F o r instance, it m a y b e t h e result o f electrostatic r e p u l s i o n , b u t also o f steric r e p u l sion. T h e s y s t e m s for w h i c h we observe relatively slow a d s o r p t i o n h a v e in c o m m o n t h a t b o t h t h e s o r b e n t surface a n d t h e p r o t e i n
m g m -2
•
•
2 - m g m -2
x r"
x 1
x
x
x
I ~.I
x
o..~°-"~ ~
1
2
I 3
,~
rain1'2
FIG. 5. Adsorption of 125I-labeledHPA as a function of t t/2 on glass (©) and siliconized glass (×) surfaces. Conditions as in Fig. 2. Initial slope calculated for a diffusion coefficient of 7.0 × 10-~ m 2 sec-~ is represented by the straight line. Journal of Colloid and Interface Science, Vol. 91, No. 1, J a n u a r y 1983
0
1
2
3
4 r a i n +lz
FIG. 6. Adsorption of 125I-labeledHPA as a function of t v2 on negatively charged polystyrene surfaces. Conditions and symbols as in Fig. 3. The straight line represents the initial slope calculated for a diffusion coefficient of 7.0 X 10-H m 2 sec-1.
ADSORPTION OF ALBUMIN
253
mg m -2
served. The reason for it may be, first, that the electrokinetic charge on the HPA molecule at pH 4 is much smaller than at pH 7.4 (3) and, second, that the albumin molecule tends to adsorb with its negative groups oriented toward the sorbent surface. The latter reason may also explain the finding that the / initial rate of adsorption of positively charged / albumin (pH 4) is slower on a negatively charged polystyrene surface than on a positive one. Since at the various surfaces, at otherwise similar conditions, the albumin molecule may undergo structural rearrangements to different extents, the steady-state values of P cannot be simply interpreted as a measure I I I I for the affinity between the protein and the 0 1 2 3 4 rain ~lz sorbent. For both the positive and the negFIG. 7. Adsorption of 125I-labeled H P A as a function ative polystyrene surface P is higher at pH of t ~/2 on positively charged polystyrene surfaces. Con4 than at pH 7.4. This is in agreement with ditions and symbols as in Fig. 4. The straight line represents the initial slope calculated for a diffusion coefalbumin adsorption at negatively charged ficient of 7.0 × 10 l~ m 2 see-L particles of, e.g., polystyrene (1), silver iodide (26), glass (27), and at the positively charged molecule are negatively charged. We are particles of hematite (28). The variation of therefore inclined to conclude that the slow F with pH has been interpreted in terms of adsorption is due to an energy barrier caused structural rearrangements in the albumin by overlapping electric fields from the neg- molecule (1-5). Although the adsorptions at ative charges on the sorbent and the protein. the positively charged polystyrene slides fit In that case, the rate of adsorption is slowed in this picture, the influence o f p H is different down by a factor e - A / k T where A is the ac- from that observed with positive polystyrene tivation energy required to overcome the bar- latices (29). With the latex the albumin adtier and k T is one unit of thermal energy. sorption at pH 7.4 is significantly larger than Hence, A can be evaluated by comparing the at pH 4. It should be realized, however, that initial slope of the curve for P (t 1/2) with the as a result of the preparation of the slides one obtained for a diffusion-controlled pro- from the latex the surface charge density may cess with D = 7.0 × 10TM m 2 sec -l. The val- be affected considerably. The influence of ionic strength has been ues thus derived for A are 2.9 k T and 0.8 k T for adsorption on untreated and siliconized studied for the adsorption on positive polyglass, respectively, both at pH 7.4 and 0.05 styrene only. At pH 4 as well as pH 7.4 adM ionic strength. For the adsorption at the sorptions are larger at higher ionic strength. negatively charged polystyrene surface at Referring to our previous studies with the 0.01 M ionic strength, the values for A are albumin-polystyrene latex systems, this ionic strength effect may be due to different degrees 1.6 k T at pH 7.4 and 0.4 k T at pH 4.0. In analogy to the above reasoning, an elec- of protein reconformation rather than to diftric energy barrier would be expected for the ferences in lateral repulsion between the adadsorption of positively charged albumin sorbed albumin molecules (1-5). The finding (pH 4) on a positively charged polystyrene that the influence of the ionic strength is surface. This, however, has not been ob- more pronounced at pH 4 than at pH 7.4,
f:
Journal of Colloid and Interface Science, Vol. 91, No. i, January 1983
254
VAN DULM AND NORDE
whereas the protein charge at pH 7.4 is larger, supports that conclusion. There is one more remarkable feature to be discussed. In some cases, namely at pH 4 and not too low ionic strength, the F(t) curves pass through a maximum at about 20 to 30 min. A few times a similar behavior has been reported in the literature. For instance, Walton and Soderquist (8) find such a maximum for the adsorption of bovine serum albumin on siliconized glass and on glass coated by a copolymer of lysine and leucine. Furthermore, Horbett (16), studying the competitive adsorption between proteins from blood plasma onto different surfaces, reports a significant desorption of albumin from a hydrophobic surface following a fast initial adsorption. He assumes that after prolonged incubation albumin is exchanged against other plasma proteins. In the experiments described in this paper as well as in Ref. (8), albumin is the only protein present, and it can, therefore, not be displaced by a different kind of protein. However, the albumin sample itself is not completely homogeneous, i.e., it may contain a small fraction of dimers and higher aggregates. Furthermore, the number of 0.7 sulfhydrylgroups per albumin molecule (30) points to the coexistence of mercaptalbumin and nonmercaptalbumin. Hence, there are a number of different species present that compete for adsorption. This could explain the adsorption maximum with time, although in that case the influence o f p H and ionic strength on the occurrence of that maximum is still to be explained. Another possibility is that desorption after some time is the result of rearrangements in the structure of already adsorbed albumin molecules. As has been mentioned in the introduction, adsorbed protein molecules may change their conformation in order to attach more segments at the sorbent surface, i.e., to optimize their surface bonding. Since the individual segments are reversibly adsorbed some protein molecules may become completely detached in favor of the "spreading" of other adsorbed moleJournal of Colloid and Interface Science, VoI.91, No. 1, January 1983
cules. Whether or not such structural rearrangements lead to some desorption depends on the relative rates of deposition of the protein on the sorbent surface and of the structural rearrangements, respectively. Thus, a maximum in P(t) is more likely to show up in cases of fast deposition and slow reconformation. Indeed, as is shown in Figs. 2-4, at pH 4 the adsorption proceeds faster than at pH 7.4. Moreover, as a result of the lower charge density, the internal coherence in the albumin molecule at pH 4 is expected to be stronger than at pH 7.4, which would imply a greater resistance to conformational changes. The absence of a peak in the F(t) curve at pH 4 and 10-3 M could then be ascribed to a reduced screening of interactions between charges at that low ionic strength. CONCLUSION
Albumin adsorption is markedly influenced by the properties of the sorbent surface. The amount of protein accommodated in the adsorbed layer is larger at a more hydrophobic surface. The adsorbed amount is furthermore determined by the ratio between the rates of deposition and the rate of structural rearrangements in the adsorbed molecule. A high value for this ratio results in higher adsorptions. The kinetics of the adsorption process depend strongly on the electric interactions. More specifically, if both the protein and the sorbent are negatively charged, the initial rate of adsorption is relatively low, whereas at other conditions it corresponds to that predicted from diffusion theory. In some cases, fast initial adsorption is followed by some desorption. This desorption of a fraction of the protein may be a consequence of progressive unfolding of the remaining adsorbed albumin molecules. ACKNOWLEDGMENT The authors gratefully acknowledge the cooperation of the Institute for the Application of Atomic Science in Agriculture, Wageningen, The Netherlands, which granted the facilities to perform the experiments involving radionuclides.
ADSORPTION OF ALBUMIN REFERENCES 1. Norde, W., and Lyklema, J., J. ColloidlnterfaceSci. 66, 257 (1978). 2. Norde, W., and Lyklema, J., J. ColloidlnterfaceSci. 66, 266 (1978). 3. Norde, W., and Lyklema, J., J. ColloidlnterfaceSci. 66, 277 (1978). 4. Norde, W., and Lyklema, J., J. Colloidlnterface Sei. 66, 286 (1978). 5. Norde, W., and Lyklema, J., Jr. ColloidlnterfaceSci. 66, 295 (1978). 6. Norde, W., and Lyklema, J., J. ColloidlnterfaeeSci. 71, 350 (1979). 7. Adams, D. J., Evans, M. T. A., Mitchell, J. R., Phillips, M. C., and Rees, P. M., J. Polym. Sci. C34, 167 (1971). 8. Walton, A. G., and Soderquist, M. E., Croatica Chem. Acta 53, 363 (1980). 9. Chan, B. M. C., and Brash, J. L., J. Colloidlnterface Sci. 84, 263 (1981). 10. Watkins, R. W., and Robertson, C. R., J. Biomed. Mater. Res. 11, 915 (1977). 11. Walton, A. G., and Maenpa, F. C., J. Colloid Interface Sci. 72, 265 (1979). 12. Gendreau, R. M., Appl. Spectrosc. 35, 353 (1981). 13. Ratner, B. D., Horbett, T. A., Shuttleworth, D., and Thomas, H. R., J. Colloid Interface Sci. 83, 630 (1981). 14. Grant, W. H., and Dehl, R. E., in "Adhesion and Adsorption of Polymers" (Lieng-Huang Lee, Ed.), Part B, p. 827. Plenum Press, New York, 1980.
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15. Brynda, E., Houska, M., Pokornh, Z., Cepalova, N. A., Moiseev, Yu. V., and Kalal, J., J. Bioeng. 2, 411 (1978). 16. Horbett, T. A., in "Adhesion and Adsorption of Polymers" (Lieng-Huang Lee, Ed.), Part B, p. 677. Plenum Press, New York, 1980. 17. Brash, J. L., and Samak, Q., J. ColloidlnterfaceSci. 65, 495 (1978). 18. Chan, B. M. C., and Brash, J. L., J. Colloidlnterface Sci. 82, 217 (1981). 19. Penners, G., Priel, Z., and Silberberg, A., 31 Colloid Interface Sci. 80, 437 (1981). 20. Furusawa, K., Norde, W., and Lyklema, J., KolloidZ. Z. Polym. 250, 908 (1972). 21. Goodwin, J. W., Ottewill, R. H., and Pelton, R., Colloid Polym. Sci. 257, 61 (1979). 22. Van der Scheer, A., Thesis, Technical University Twente, The Netherlands, 1978. 23. MacRitchie, F., ,L Colloid Interface Sci. 38, 484 (1972). 24. Brash, J. L., and Davidson, V. J., Thrornb. Res. 9, 249 (1976). 25. Keller, K. H., Canales, E. R., and Yum, S. I., J. Phys. Chem. 75, 379 (1971). 26. Matuszewska, B., Norde, W., and Lyklema, J., Z Colloid Interface Sci. 84, 403 (1981). 27. Hull, H. B., Biochim. Biophys. Acta 19, 464 (1956). 28. Koutsoukos, P., Lyklema, J., and Norde, W., J. Colloid Interface Sci., submitted. 29. Koutsoukos, P., Mumme-Young, C. A., Norde, W., and Lyklema, J., Colloids and Surfaces, 5, 93 (1982). 30. Andersson, L., Biochim. Biophys. Acta 117, 115 (1966).
Journal of Colloid and Interface Science, Vol. 91, No. 1, January 1983