Volumetric interpretation of protein adsorption: Kinetics of protein-adsorption competition from binary solution

Abstract

The standard solution-depletion method is implemented with SDS-gel electrophoresis as a multiplexing, separation-and-quantification tool to measure competition between two proteins (i and j) for adsorption to the same hydrophobic adsorbent particles (either octyl sepharose or silanized glass) immersed in binary-protein solutions. Adsorption kinetics reveal an unanticipated slow protein-size-dependent competition that controls steady-state adsorption selectivity. Two sequential pseudo-steady-state adsorption regimes (State 1 and State 2) are frequently observed depending on i, j solution concentrations. State 1 and State 2 are connected by a smooth transition, giving rise to sigmoidally-shaped adsorption-kinetic profiles with a downward inflection near 60 minutes of solution/adsorbent contact. Mass ratio of adsorbed i, j proteins (m i m j ) remains nearly constant between States 1 and 2, even though both m i and m j decrease in the transition between states. State 2 is shown to be stable for 24 hours of continuous-adsorbent contact with stagnant solution whereas State 2 is eliminated by continuous mixing of adsorbent with solution. In sharp contrast to binarycompetition results, adsorption to hydrophobic adsorbent particles from single-protein solutions (pure i or j) exhibits no detectable kinetics within the timeframe of experiment from either stagnant or continuously-mixed solution, quickly achieving a single steady-state value in proportion to solution concentration. Comparison of binary competition between dissimilarly-sized protein pairs chosen to span a broad molecular-weight (MW) range demonstrates that selectivity between i and j scales with MW ratio that is proportional to protein-volume ratio (ubiquitin, Ub, MW = 10.7 kDa; human serum albumin, HSA, MW = 66.3 kDa; prothrombin, FII, 72 kDa; immunoglobulin G, IgG, MW = 160 kDa; fibrinogen, Fib, MW = 341 kDa). Results are interpreted in terms of a kinetic model of adsorption that has protein molecules rapidly diffusing into an inflating interphase that is spontaneously formed by bringing a protein solution into contact with a physical surface (State 1). State 2 follows by rearrangement of proteins within this interphase to achieve the maximum interphase concentration (dictated by energetics of interphase dehydration) within the thinnest (lowest volume) interphase possible by ejection of interphase water and initially-adsorbed proteins. Implications for understanding biocompatibility are discussed using a computational example relevant to the problem of blood-plasma coagulation.

A review of protein adsorption on bioceramics

“…Another interesting aspect of protein adsorption to bioceramics is competition adsorption between two or more proteins on the same adsorbent surface [223][224][225]. Leo Vroman firstly observed that fibrinogen preferentially adsorbed onto tantalum surfaces from blood plasma.…”

“…However, certain proteins, such as human serum albumin, are observed to be relatively resistant to displacement at hydrophobic surfaces. The competitive adsorption of proteins is highly dependent on the concentrations and competing components [223,226]. Brash & Lyman [229] demonstrated that protein adsorption is in direct proportion to the protein concentration.…”

Fibrinogen adsorption on blocked surface of albumin

“…Even though protein monolayer adsorption is a common hypothesis within the community of protein adsorption and biomaterials, there are also studies arguing for the possibility of proteins adsorbing in a multilayer fashion [9,15,[33][34][35]. Results from experiments with radioactive labelled proteins suggest that the hydrophobic PET surface can induce large conformational changes in adsorbed proteins and that those changes can induce enhanced adsorption of later arriving proteins onto the surface [24].…”

Understanding and modulating the competitive surface-adsorption of proteins through coarse-grained molecular dynamics simulations

“…However, the mechanisms of the phenomenon are still debated and no existing model can fully explain it 17,22,23 .…”

“…Volumetric effects, due to non-deformable proteins trying to fit on the available surface, can account for competitive adsorption of proteins 17 . However, they do not reproduce the maxima of absorption of the Vroman effect.…”