Kinetics and Thermodynamics of Protein Adsorption: A GeneralizedMolecular Theoretical ApproachFang Fang and Igal SzleiferDepartment of Chemistry, Purdue University, West Lafayette, Indiana 47907 USAABSTRACT The thermodynamics and kinetics of protein adsorption are studied using a molecular theoretical approach. Thecases studied include competitive adsorption from mixtures and the effect of conformational changes upon adsorption. Thekinetic theory is based on a generalized diffusion equation in which the driving force for motion is the gradient of chemicalpotentials of the proteins. The time-dependent chemical potentials, as well as the equilibrium behavior of the system, areobtained using a molecular mean-field theory. The theory provides, within the same theoretical formulation, the diffusion andthe kinetic (activated) controlled regimes. By separation of ideal and nonideal contributions to the chemical potential, theequation of motion shows a purely diffusive part and the motion of the particles in the potential of mean force resulting fromthe intermolecular interactions. The theory enables the calculation of the time-dependent surface coverage of proteins, thedynamic surface tension, and the structure of the adsorbed layer in contact with the approaching proteins. For the case ofcompetitive adsorption from a solution containing a mixture of large and small proteins, a variety of different adsorptionpatterns are observed depending upon the bulk composition, the strength of the interaction between the particles, and thesurface and size of the proteins. It is found that the experimentally observed Vroman sequence is predicted in the case thatthe bulk solution is at a composition with an excess of the small protein, and that the interaction between the large proteinand the surface is much larger than that of the smaller protein. The effect of surface conformational changes of the adsorbedproteins in the time-dependent adsorption is studied in detail. The theory predicts regimes of constant density and dynamicsurface tension that are long lived but are only intermediates before the final approach to equilibrium. The implications of thefindings to the interpretation of experimental observations is discussed.INTRODUCTIONProtein adsorption plays a major role in a variety of impor-tant technological and biological processes (Clerc and Lu-kosz, 1997; Denizli et al., 2000; Ghose and Chase, 2000;Hlady and Buijs, 1996; Montdargent and Letourneur, 2000;Shi and Ratner, 2000; Slomkowski, 1998; Topoglidis et al.,1998). For example, blood proteins tend to adsorb intosurfaces of foreign materials. This is the first step on sur-face-induced thrombosis (Andrade and Hlady, 1986; Hor-bett, 1993; E. F. and S. 1993; Tanaka et al. 2000). A largenumber of biotechnological devices include surface-boundproteins, e.g., biosensors (Nyquist et al., 2000; Slomkowskiet al., 1996; Sukhishvili and Granick, 1999; Zhou et al.,2000). Separation of proteins by chromatography involvesthe competitive adsorption of the particles (Wang 1993).The understanding of the fundamental factors that deter-mine protein adsorption are imperative to improve our abil-ity to design biocompatible materials and biotechnologicaldevices. Moreover, protein adsorption is a very importantfundamental problem that involves large competing energyscales and conformational statistics that may result in re-versible and irreversible processes.The adsorption of proteins on surfaces is a complexprocess. The adsorbing particles are large, and, thus, thesurface–protein interactions are usually long range and thestrength is many times the thermal energy. Further, due tothe large size and the shape of the particles, the interactionsbetween the adsorbed particles on the surface are nontrivialand can be strongly influentiated by the fact that the parti-cles may undergo conformational changes upon adsorption(Billsten et al., 1995; Ishihara et al., 1998; Kondo andFukuda, 1998; Nasir and McGuire, 1998; Norde and Gia-comelli, 1999, 2000; Tan and Martic, 1990; Van Tassel etal., 1998; Gidalevitz et al., 1999). Actually, the kinetics andthermodynamics of protein conformational changes on thesurface is a very complex subject and their understanding isat its early stages. The idea behind the work presented hereis an attempt to formulate a molecular theoretical approachthat can be applied to study both the equilibrium and thekinetic behavior of protein adsorption.On experimental studies (Green et al., 1999; Malmsten,1997), it has been observed that, when two or more kinds ofproteins are present in solution, such as in blood plasma, theadsorption is the result of the competition between the timescale to reach the surface and the strength of the surface–protein interaction. For example, in blood plasma solutionsof albumin, immunoglobulin-G (IgG) and fibrinogen (Fgn)in contact with a polystyrene surface, the initial adsorptionis dominated by the smaller protein (albumin), which arealso at larger concentrations in the bulk, to be later replacedby the larger proteins like IgG and Fgn. This sequentialadsorption is called the Vroman sequence. In other experi-Received for publication 13 November 2000 and in final form 22 March2001.Address reprint requests to Igal Szleifer, Purdue University, Dept. ofChemistry, Brown Building 1393, West Lafayette, IN 47907-1393. Tel.:765-494-5255; Fax: 765-494-0239; E-mail: [email protected].© 2001 by the Biophysical Society0006-3495/01/06/2568/22 $2.002568 Biophysical Journal Volume 80 June 2001 2568 –2589ments (Lassen and Malmsten, 1997), different adsorptionpatterns are observed when the surfaces are changed. On thehydrophobic PP-HMDSO (hexamethyldisiloxane), surfacealbumin and IgG dominate the adsorption. However, onhydrophilic PP-DACH (1,2-diaminocyclohexane) andPP-AA (acrylic acid) surfaces, Fgn is almost exclusivelyfound on the surface. These experimental observations dem-onstrate that the incorporation of the solution conditions andthe protein–surface interactions have to be considered forthe proper understanding and description of the adsorptionprocess.One of the most important contributions to the under-standing of the kinetics of protein adsorption is the randomsequential adsorption (RSA) model (Feder and Giaever,1980; Schaaf and Talbot, 1989). In this approach, the pro-teins are assumed to be rigid particles that interact onlythrough excluded volume interactions. The particles areassumed to irreversibly adsorb to the