ProteinProtein Interactions Affecting Proteins at Surfaces - American

Chapter 12

Protein—Protein Interactions Affecting Proteins at Surfaces Peter H. Warkentin, Ingemar Lundström, and Pentti Tengvall

Downloaded by UNIV LAVAL on April 24, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch012

Biomaterials Consortium, Laboratory of Applied Physics, Linköping University, S-581 83 Linköping, Sweden

Protein adsorption to methylated silicon gradient and hydrophilic silicon surfaces as single, binary, and tertiary purified components as well as in association with normal plasma were studied. Antibody and albumin incubations of protein and plasma treated surfaces were noted to affect ellipsometric thickness and antibody binding patterns. BSA blocking procedures on surfaces treated with pure fibrinogen were able to mimic, to a degree, the same ellipsometric patterns as plasma treated gradients when probed by specific antibodies. Indications are that serum albumin influences and may even participate in the removal of IgG and fibrinogen from certain surfaces, even as a subsequent, non-competitive step. HMWK adsorbs differently to surfaces dependent on whether it is a serum component or a single protein. Specific antibody incubations of protein and plasma treated surfaces have been observed to cause a reduction in the ellipsometrically observable surface bound protein layer. These relationships may be due to components other than those indicated by the Vroman sequence. Protein adsorption at the blood-material interface is a complex interaction that has yet to be thoroughly defined. It is known to be a function of the surface with which proteins interact but it is also true that protein-surface associations are not the same in mixed protein situations as in pure protein solutions. Increasingly we have had to address the fact that proteins respond not only to the surfaces that they have been introduced to but also each other. In the following studies we see how proteins compete in limited systems and how this behaviour differs if the competitive edge is removed. In a previous paper (1), we have presented studies showing that protein-protein interactions affect protein interactions towards the surface in limited protein systems. These surface interactions vary according to the order and combination of the other proteins involved in the experimental model. Previous findings by Deyme et al (2) and Baszkin and Boissonade (3) indicate similar interactive behaviour of proteins where the presence of collagen resulted in increased albumin adsorption to solution-air and solution-polyethylene interfaces. These researchers support the approach of the study of limited defined protein systems stating that "detailed studies of binary solutions must be carried out with consideration to both components in such a system". It is through studies of simple systems such as these that the behaviour of more complex protein mixtures such as blood serum and blood plasma can be better understood.

0097-6156/95/0602-0163$12.00/0 © 1995 American Chemical Society Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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This paper offers further evidence of protein-protein interactions towards surfaces focusing largely, but not exclusively, on albumin affects. Plasma and serum system experiments illustrate that while these complex mixtures do not give results identical to those observed in defined protein systems, there are effects produced by the introduction of albumin that result in similar protein detection patterns. While there have been many models presented to explain protein adsorption onto surfaces they are often dependent on proteins behaving strictly as "particles" bearing average physical characteristics that have been verified. Some of these particle properties are however assigned for the convenience of the model (4). Other researchers have offered models of more complex proteins by partitioning physical characteristics thereby allowing differential behaviour by the particle model (5). There is also a tendency to regard the model particle only with respect to the material interface. While these assumptions aid the model, they tend to ignore subtle interactions and differences in real proteins that may limit the models applicability especially in real world multi-component systems. In order to study variable surface characteristics we have often employed a methylation gradient over a negatively charged hydrophilic silicon surface (6) such that the degree of surface energy can be modified in a controlled manner. This results in a surface that is extremely hydrophobic at one end (water contact angle >85°) to very hydrophilic (water contact angle 95% of the protein. The human IgG used was a commercial gammaglobulin fraction from KabiVitrum A B , Sweden of a quality suitable for human injection. The source of purified high molecular weight kininogen (HMWK) used in gradient studies was Calbiochem,

Horbett and Brash; Proteins at Interfaces II ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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WARKENTIN ET AL.

U.S.A. while discrete surface energy studies were performed using a purified single chain high molecular weight kininogen obtained from Enzyme Research Laboratories, U.S.A. Citrated human plasma was pooled from five apparently healthy donors. H M W K deficient plasma was obtained from Sigma as lyophilized powder which was reconstituted immediately before use with deionized water. Rabbit anti-human IgG, rabbit anti-human albumin, rabbit anti-human fibrinogen, and alkaline phosphatase conjugated swine anti-rabbit IgG were all polyclonal IgG fractions from Dakopatts, Denmark. Rabbit anti-human H M W K was a polyclonal IgG fraction from Calbiochem while the goat anti-human H M W K antiserum was purchased from Nordic, Finland and an alternate goat anti-human H M W K antiserum was obtained as a gift from Enzyme Research Laboratories, U.S.A. Commercially produced Type P(100) boron doped silicon wafers with a diameter of 76.2 ± 0.5 mm were obtained from Okmetic, Finland, cut to required sizes (1 cm x 1 cm) and used in hydrophilic and hydrophobic flat surface experiments. Silicon gradient surfaces, 10 mm x 37 mm, were also cut from the same silicon wafers. Ellipsometry. The ellipsometric one-zone measurements were made on dried protein surfaces, and using a Rudolph Research Auto El EI ellipsometer. The protein concentration was then estimated assuming a constant film refractive index no = nf= 1.465 according to the method of Stenberg and Nygren (9), X

2

Surface concentration (ng/mm ) « K x thickness (nm) where K is the density of the protein « 1.35 g/1. Since the refractive index of proteins, n « 1.55, the thickness of the protein film is decreased by a factor: p

2

2

2

[ (nVl)/n ox ] / [ (n p-i)Ai p ]

thus giving K « 1.2 (9)

As the refractive indices, nf, may vary slightly for different proteins and on different substrates, the calculated values reported in this paper are effective values on silicon and DDS-methylated silicon, respectively.The error bars presented in the graphs indicate the statistical errors of the measured values at the surface. They do not include systematic discrepancies due to the approximations introduced by the refractive indices or mathematical modelling techniques. A previous comparison of in situ ellipsometric and radiotracer experimental values of adsorbed amounts of proteins onto silicon surfaces demonstrate reasonable agreement between the two techniques (10). Silicon Surface Protein Treatments. Wettability gradients were produced on silicon surfaces using the method of Elwing et al (6). Silicon wafers were cut into sizes of 10 x 37 mm and made hydrophilic (water contact angle < 5°) by heating to 80°C in a 1:1:5 (volume) solution of NH3:H2O2:H20 (deionized). They were washed 3 times in deionized water heated to 80°C in a 1:1:6 solution (volume) of HC1:H202:H20 (deionized) and finally washed 3 times in deionized water and stored until use in acidified H 2 O . These were placed vertically in a container filled with xylene and a 0.05% solution of DDS in trichloroethylene which was carefully layered below the xylene phase and permitted the DDS to diffuse into the upper phase for 90 minutes. The contents were then drained from the bottom and the surfaces washed sequentially with ethanol, trichloroethylene, and ethanol and finally dried under nitrogen stream prior to use. Depending on the particular experiment, proteins were either used at human physiological concentrations or brought to 1/10 physiological concentrations in PBS as indicated in the review of Andrade and Hlady (11) or as indicated by Miiller-Esterl (12)




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with respect to human H M W K . Some researchers have, in the past, chosen to use Tris buffered saline to overcome the problem of phosphate interfering with absorption of some proteins, particularly fibrinogen (13). On the other hand, Tris buffers are less physiological and, when used in adsorption steps, interfered with H M W K detectability (14). Since it has never been established how the bias introduced by the use of various buffer differs from the absolute physiological adsorption conditions, we use PBS as a standard buffer. Physiological concentrations were uniformly defined as 12.5 mg/ml for IgG, 2.5 mg/ml for fibrinogen and 40 mg/ml for albumin. H M W K was diluted to final physiological concentration of 0.074 mg/ml. Plasma was diluted 1:9 in PBS for comparisons with the single protein solutions where indicated. Incubation times are either 10 minutes or 1 hour depending on the experiment. In B S A blocked plasma experiments, citrated plasma diluted to 10% physiological in PBS was incubated for 1 hour followed by 1 hour in 1% (10 mg/ml) B S A in PBS. In experiments using BSA, H M W K , and plasma treated gradients, duplicate surfaces were used and representative surfaces are shown in the figures. In all other experiments triplicates were used and results are represented statistically. Experimental results were corrected for background DDS methylation and oxide layer by measuring untreated surfaces and subtracting these values. Gradients were washed with PBS and incubated in a 1.8 ml solution per gradient in either the plasma or protein solution for 1 hour. In experiments using discrete surfaces, 3 surfaces of 1 cm2 were incubated in 1.8 ml. Surfaces were washed in PBS followed by deionized water, dried under nitrogen and the initial layer read by ellipsometry. In blocking experiments surfaces were treated as above except that they were blocked for 1 hour in 1% BSA after exposure to the initial protein treatment and rinsed in PBS and deionized water prior to the first ellipsometric reading. In controls and other experiments where surfaces were checked for amplified secondary antibody binding or non-specific antibody adsorption they were incubated in swine anti-rabbit IgG-alkaline phosphatase, 1/50 dilution in a PBS solution for 1 hour and washed in TBS followed by water, dried under nitrogen and read again. The conjugated antibody was used to keep the reagent consistent with previous ELISA and surface imaging work (14). Where there was primary antibody binding the surfaces were checked for further specific ellipsometric increases while in the case of no specific primary antibody incubation, they indicate only non-specific background binding. BSA Treatment of Protein Adsorbed Gradients. Gradients were incubated with IgG, 1.25 mg/ml or fibrinogen, 0.25 mg/ml in PBS for 1 hour. Half the number of surfaces were blocked for 1 hour in 1% B S A in PBS and read by ellipsometry. A l l surfaces, blocked and non blocked, were incubated in duplicate for 1 hour at room temperature in 1.8 ml/surface of the following antibody solutions diluted 1/50 in PBS: rabbit anti-human IgG, rabbit anti-human fibrinogen, and rabbit anti-human H M W K . Surfaces were then washed in PBS followed by deionized water, dried under nitrogen and read by ellipsometry. These represented surfaces after primary antibody incubation. The surfaces were then washed in PBS and incubated for 1 hour at room temperature in a 1/50 PBS solution of swine anti-rabbit IgG-alkaline phosphatase as secondary antibody. After the second antibody treatment, surfaces were washed 3 times in TBS and rinsed in deionized water. The surface thickness was read after every incubation step by ellipsometry. Results and Discussion Plasma Treated Gradients. Plasma treated gradient surfaces displayed a fair degree of variation in surface thicknesses as measured by ellipsometry (± 1.9 ng/mm , not shown). It is not certain whether this is due to variability in gradient preparation or 2


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Protein-Protein Interactions

changes in plasma protein adsorption. The differences in plasma treated surfaces alone and surfaces plasma treated followed by B S A blocking, however, were not statistically conclusive (not shown). When the same surfaces are incubated with the amplifying antibodies alone there was no alteration in thickness indicating that the secondary antibodies in the absence of specific primary antibodies did not add to ellipsometric thickness. In surfaces which were both treated with plasma alone (not shown) and those followed by B S A blocking (Fig. 1), there was no statistically conclusive indication that rabbit anti-IgG altered in its ability to bind the surface adsorbed IgG, although there appeared to be a reduction from 8.4 ng/mm without B S A to 7.2 ng/mm with B S A on the hydrophobic end. In surfaces which were both treated with plasma alone and those which were blocked, the anti-fibrinogen demonstrated a maximal ellipsometric thickness subterminally with respect to the hydrophobic end. When B S A incubations are introduced prior to primary antibody treatment there is a significant reduction in thickness of about 4.8 ng/mm from peak maxima. This reduction is not as great at the hydrophilic end of plasma treated surfaces but is still noticeable. 2

2


2

Single Protein Treated Gradients. The single protein adsorption profiles are in agreement with previous observations (6, 1). IgG treated surfaces gave a smooth decrease in thickness along the gradient from an average of 5.0 ng/mm at the hydrophobic side to 1.9 ng/mm at the hydrophilic side (Fig. 2a). On B S A blocked surfaces this pattern is much the same, 4.8 ng/mm to 2.5 ng/mm (Fig. 2b). Primary antibody incubation of surfaces treated with IgG in the absence of B S A blocking (Fig. 2a) gives an average increase in thickness to 12.1 ng/mm at the hydrophobic end to 10.1 ng/mm at the hydrophilic end. This indicates confluent, albeit differential, IgG binding across the entire gradient. Surfaces that were exposed to B S A after IgG incubations display a remarkably different pattern however upon primary antibody incubation. The thickness at the hydrophobic end is unchanged (12.1 ng/mm ) but very much lower (2.5 ng/mm ) at the hydrophilic end (Fig. 2b). This drop-off is most pronounced from 5 to 11 mm from the hydrophobic end. The B S A in this case has definitely affected the preexisting IgG layer, either by removing and/or replacing it or by compromising its antigenicity. The degree of the B S A influence is a function of the surface characteristics, as well as the protein on the surface, otherwise IgG across the entire gradient would have been affected to the same extent. Human serum albumin in plasma may act in the same manner. Although it is in lower concentration than is these blocking experiments, it may be more competitive since it is in solution at the same time as IgG as opposed to the post-incubation exposure of B S A in this experimental model. The pure fibrinogen story on gradients resembles IgG in may ways. As a pure protein on the gradient (Fig. 3a) it resides as two distinct ellipsometric thicknesses, one at the hydrophobic end of 4.9 ng/mm and one at the hydrophilic end of 3.5 ng/mm From 8 to 12 mm from the hydrophobic end there is a steeper transition in thickness than at the two extremes. The B S A blocked profile (Fig. 3b) is essentially the same displaying no apparent protein thickness changes. Upon application of the rabbit antifibrinogen there is a confluent shift upward of the entire fibrinogen profile to 14.5 ng/mm at the hydrophobic end and 12.5 ng/mm at the hydrophilic end (Fig. 3a). In the B S A blocked surfaces (Fig. 3b) the thickness is submaximal at the hydrophobic end and increases to a maximum again at a position 3 mm from the hydrophobic terminus. This low terminal antigenicity is similar to the B S A blocked or unblocked plasma and not noted in anti-fibrinogen activity where albumin is not a component. It may be a direct effect of the presence of albumin. The anti-fibrinogen remains maximal and begins to drop at a distance 8-9 mm along the gradient which reflects the transition point of the fibrinogen alone. The drop in antibody binding thickness at this point is similar to the effect on IgG-anti-IgG treated surfaces but in this case the drop in thickness is not as great. The pattern is similar to the anti-fibrinogen binding to B S A 2

2

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2

2

2

2

2

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Plasma —>RbaFib Plasma — >BSA—>RbctFib Plasma—>BS A

0 RbalgG

14.0 12.0 10.0 8.0


6.04.0 IgG 2.0 0.0

0-90°

0=45° 10

0

0 BSA—> RbalgG

*—
9O° 0

0

ProteinProtein Interactions Affecting Proteins at Surfaces - American

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