Volumetric interpretation of protein adsorption: Competition from mixtures and the Vroman effect

Abstract

A Vroman-like exchange of different proteins adsorbing from a concentrated mixture to the same hydrophobic adsorbent surface is shown to arise naturally from the selective pressure imposed by a fixed interfacial-concentration capacity (w/v, mg/mL) for which protein molecules compete. A size (molecular weight, MW) discrimination results because fewer large proteins are required to accumulate an interfacial w/v concentration equal to smaller proteins. Hence, the surface region becomes dominated by smaller proteins on a number-or-mole basis through a purely-physical process that is essentially unrelated to protein biochemistry. Under certain conditions, this size discrimination can be amplified by the natural variation in protein-adsorption avidity (quantified by partition coefficients P) because smaller proteins (MW <50 kDa) have been found to exhibit characteristicallyhigher P than larger proteins (MW >50kDa). The standard depletion method is implemented to measure protein-adsorption competition between two different test proteins (i and j) for the same hydrophobic octyl sepharose adsorbent particles. SDS-gel electrophoresis is used as a multiplexing, separation-and-quantification tool for this purpose. Identical results obtained using sequential and simultaneous competition of human immunoglobulin G (IgG, protein j) with human serum albumin (HSA, protein i) demonstrates that HSA was not irreversibly adsorbed to octyl sepharose over a broad range of competing solution concentrations. A clearly-observed exchange of HSA for IgG or fibrinogen (Fib) shows that adsorption of different proteins (i competing with j) to the same hydrophobic surface is coupled whereas adsorption among identical proteins (i or j adsorbing from purified solution) is not coupled. Interpretive theory shows that this adsorption coupling is due to competition for the fixed surface capacity. Theory is extended to hypothetical ternary mixtures using a computational experiment that illustrates the profound impact size-discrimination has on adsorption from complex mixtures such as blood.

Volumetric interpretation of protein adsorption: Kinetic consequences of a slowly-concentrating interphase

“…Understanding how protein arrives at, and adsorbs to, biomaterial surfaces is thus one of the most fundamental problems of biomaterials surface science. It is of practical interest therefore, to consider how this work might impact understanding of the fundamentals of biocompatibility.Based on the findings of this and recent work on protein adsorption [22][23][24]38], we conclude that immersion of a hydrophobic surface into a concentrated, multi-component protein solution such as blood [78,79] leads to virtually instantaneous accumulation of protein molecules from the proximal fluid phase (relative to the timescale of the observable acute biological responses to materials; see Appendix A). Given that (i) surface capacity for protein is actually quite small (in the range of 2-3 mg/m 2 or µmoles/m 2 for kDa-size proteins), (ii) the total blood-protein concentration is large (50-60 mg/mL, [78,79]), and that (iii) proteins exhibit surprisingly little difference in adsorption energetics across a broad range of blood protein types (3 decades in molecular weight, see refs.…”

“…Solution of the fundamental surface thermodynamic adsorption Eq. (4) in terms in terms of time-dependent interphase concentration C I (t) for dilute solutions predicts that protein adsorption at equilibrium should be governed by a partition coefficient P, as has been observed experimentally for various blood proteins adsorbing to surfaces spanning a full range of water wettability [22][23][24]. Protein-adsorption experiments that do not comply with such a partitioning process must thus fall outside the range of applicability of the fundamental adsorption equation and, conversely, protein-adsorption experiments exhibiting such behavior probably approximate conditions contemplated by reversible thermodynamics, including reversible partition of protein between interphase and bulk solution [9,21].…”

“…The Random Sequential Adsorption (RSA) model [14][15][16], and the venerable Langmuir adsorption isotherm [17][18][19][20] from which RSA models ultimately originate, are implicitly energy-barrier models as well. Here, net adsorption is controlled by opposing adsorption/desorption rates which, according to activated-rate theory, are moderated by activation energy barriers, with lower barriers corresponding to faster rates.Our recent work has focused on energy-and-mass-balance in protein adsorption from stagnant solutions [5][6][7][8]10,[21][22][23][24][25][26], motivated by the idea that these complimentary measures of adsorption for a broad range of proteins should clarify unresolved mechanistic issues such as adsorption reversibility, formation of adsorbed multilayers, and applicability of thermodynamic models. This work has emphasized that adsorption is a partitioning process [27,28] distributing both protein and water molecules between the bulk-solution phase and a three-dimensional (3D) interphase region (Guggenheim surface construction [28,29]).…”

“…In other words, the interface (interphase) should properly regarded as a volume comprised of both 2D surface and subsurface region -not just 2D surface alone. This distinction between 2D and 3D becomes particularly important in the event of multilayer adsorption, as has been shown to occur for proteins by a number of investigators using a variety of experimental methods over the last twenty years or so [5,6,10,22,24,26,[55][56][57][58][59][60][61][62][63][64].A primary issue with the 2D model applied to the interpretation of principal observations (i) and (ii) of the preceding section is that it eliminates an important degree-of-freedom that allows adsorbate concentration to vary independent of adsorbed mass. Partly because of this, we suspect, modern embellishments of RSA theory are forced to embrace an ever-increasing number of variables to explain all experimental outcomes (see, as a recent example, ref.…”

Vitronectin as a Micromanager of Cell Response in Material‐Driven Fibronectin Nanonetworks

“…Furthermore, VN acts as a nucleation point for the adsorption of FN molecules, as exemplified by adsorbing FN onto preadsorbed clusters of VN molecules; the resulting large protein fibrils have similar lengths to the ones achieved through co‐adsorption and include most of the VN aggregates (Figure 2). Thus, we speculate that VN acts similarly during the initial stages of adsorption from a mixture of the proteins, which are known to be dominated by the species of lower molecular weight 33. As part of the ensuing protein network, VN molecules appear to gain higher exposure when adsorbed onto PEA in the presence of FN compared to the aggregates formed upon adsorption from a pure VN solution (Figure 1E); interestingly, available molecules are preferentially located in the branch points of the matrix (Figure 3), pointing again at their activity as nucleation points.…”