44523-AC5
Protein Adsorption from Aqueous Solution with Relevance to Fouling
The overarching hypothesis of the proposal was “the hydrophobic effect dominates protein adsorption from aqueous-buffer solution to surfaces spanning the full range of water wettability”. This hypothesis was to be tested using a novel implementation of the solution-depletion method to accomplish three specific aims: (1) Measure partition coefficients as a function of adsorbent surface energy (water wettability) for a broad array of purified proteins; (2) Correlate interphase volume occupied by purified proteins with molecular dimensions, deduce interphase thicknesses/number of layers occupied by adsorbed protein, and follow changes in interphase volume as a function of adsorbent surface energy; and (3) Systematically examine the role of protein size in adsorption competition and evaluate how specific interactions among proteins (e.g. antibody-antigen interactions) affect measured adsorption competition.
As reported in 2007, three key publications substantially accomplished these aims in a manner that supported the overarching hypothesis. In brief summary, supported work shows how adsorbent surface energy controls adsorption capacity by controlling the energy required to displace interfacial water and illuminates how protein can adsorb selectively from multi-component mixtures.
Aims 2 and 3 remained to be completed by work in 2008 for which a no-cost extension was granted. The extension was needed to so that a post-doctoral fellow could be hired to pursue work of a graduated doctoral student (Hyeran Noh). A postdoctoral fellow was hired (Purnendu Parhi).
Using techniques developed in the preceding reporting period toward completion of Aims 2 and 3, we measured rate of mass adsorption from solution and compared this to the rate of decrease in interfacial energetics due to adsorption. We discovered that interfacial tensions of protein-containing solutions decreased slowly over the first hour to a steady-state value while, over this same period, the total adsorbed-protein mass was constant (for lysozyme, 15 kDa; albumin, 66 kDa; prothrombin, 72 kDa; IgG, 160 kDa; fibrinogen, 341 kDa studied in this work). These seemingly divergent observations were rationalized by the fact that interfacial energetics (tensions) are explicit functions of solute chemical potential (concentration), not adsorbed mass. Hence, rates-of-interfacial-tension-change paralleled a slow interphase concentration effect whereas solution depletion detected a constant interphase composition within the time frame of experiment. A straightforward mathematical model approximating the perceived physical situation led to an analytic formulation that was used to compute time-varying interphase volume and protein concentration from experimentally-measured interfacial tensions. Derivation from the fundamental thermodynamic adsorption equation verified that protein adsorption from dilute solution is controlled by a partition coefficient at equilibrium, as is observed experimentally at steady state. Results have been published in the journal Biomaterials.
It was further discovered that adsorption competition kinetics between two different test proteins (i and j ) for the same hydrophobic octyl sepharose (OS) adsorbent particles immersed in binary aqueous-buffer solutions exhibited two sequential pseudo-steady-state adsorption regimes (State 1 and State 2) connected by a smooth transition, giving rise to sigmoidally-shaped adsorption-kinetic profiles. State 2 was shown to be stable for 24 hours of continuous solution/adsorbent contact. Mass ratio of adsorbed i, j proteins remained constant between States 1 and 2, even though both masses decreased in the transition between states. Comparison of kinetic competition between human serum albumin as protein i (HSA, MW = 66 kDa) and j proteins selected from among ubiquitin (Ub, MW = 10.7 kDa), immunoglobulin G (IgG, MW = 160 kDa), or fibrinogen (Fib, MW = 341 kDa) demonstrated that competition between i and j scales with relative MW (proportional to relative protein volume). This outcome strongly suggests that interfacial packing is a controlling factor that determines relative adsorbed masses. Results were further interpreted in terms of the kinetic model of adsorption described above. Accordingly, protein molecules rapidly diffuse into an inflating 3D surface region (a.k.a. interphase) 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 initially-adsorbed proteins. The State 1 to State 2 transition was found to be blurred by continuous mixing of protein solution and adsorbent, giving rise to erratic adsorption measurements that more strongly resemble State 1 than State 2. Sensitivity of adsorption competition to mixing suggests there is a delicate exchange dynamic between the interphase and bulk solution that mixing perturbs, preventing achievement of the maximally-packed State 2 interphase. These results are being prepared for publication. It is unclear if the final part of aim 3 (how specific interactions among proteins affect measured adsorption competition) is technically feasible at this time given the complexity of adsorption competition between two proteins with no specific interactions.
