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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.  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) and work toward completion of proposed aims ensued.

A fourth publication in the journal Biomaterials developed a mathematical model to compare rates of mass adsorption to rates-of-interfacial-tension-change due to adsorption for the case of single purified proteins adsorbing from solution to hydrophobic surfaces.  As briefly summarized in the 2008 report, this work showed that mass adsorption was much faster than change in interfacial tension.  Time-dependent change in interfacial tension was thus shown not to be due to increasing amount of protein adsorbed to the surface, as conventionally thought, but rather due to concentration of protein within the surface region by loss of interfacial water.  These observations were pursued in the latter half of 2008 and finalized in this reporting period by examining kinetics of adsorption competition between two proteins in solution for adsorption to the same hydrophobic surface (aim #3).  This very detailed work, now published in the journal Biomaterials (paper #5), revealed an unanticipated slow protein-size-dependent competition that controlled steady-state adsorption selectivity between competing proteins. Two sequential pseudo-steady-state adsorption regimes (State 1 and State 2) were frequently observed depending on i, j solution concentrations.  State 1 and State 2 were connected by a smooth transition, giving rise to sigmoidally-shaped adsorption-kinetic profiles with a downward inflection near 60 minutes of solution/adsorbent contact.  In sharp contrast to binary-competition results, adsorption to hydrophobic adsorbent particles from single-protein solutions exhibited no detectable kinetics within the timeframe of experiment from either stagnant or continuously-mixed solution. Results were 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.  Thus it became clear that the final part of aim 3 (how specific interactions among proteins affect measured adsorption competition) is not technically feasible at this time because of the complexity of adsorption competition between two proteins with no specific interactions.

In lieu of pursuing the final part of aim 3, we worked toward resolution of an open issue related to aim 2 revealed by work outlined above.  By comparing adsorption from single- and binary-protein solutions, we discovered that protein can, and in many cases must, adsorb in multiple layers.  This seemingly incontrovertible evidence of multilayer adsorption is of considerable importance because many extant models of protein adsorption are premised on the idea that protein adsorption is due to strong interactions between protein and surface that lead to an adsorbed monolayer.  Experimental observation of multiple adsorbed layers is completely inconsistent with such theories because proteins within second (or higher-order) layers are too distant from the adsorbent surface to be held surface bound by interaction forces in close proximity.  We measured silanized-glass-particle adsorbent capacities from full adsorption isotherms of human serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa).  Adsorbent capacity expressed as either mass-or-moles per-unit-adsorbent-area increased with protein molecular weight (MW) in a manner that was shown to be quantitatively inconsistent with the idea that proteins adsorb as a monolayer at the solution-material interface in any physically-realizable configuration or state of denaturation.  Multilayer adsorption accounts for adsorbent capacity over-and-above monolayer.  This final part of supported work has been accepted for publication in the journal Biomaterials (publication #6) and, at this writing, has appeared on-line.

This final report closes PRF-funded research against specific aims of the originating proposal.  The PI is grateful for PRF support.