Statistical thermodynamic and kinetic models have been developed to describe rates and equilibria of reversible adsorption of proteins to surfaces under conditions such that a particular protein may adsorb in more than one conformation. The results of kinetic simulations suggest that early binding of conformations that are energetically favored at low surface occupancy, but ultimately entropically disfavored at high surface occupancy, may result in a substantial kinetic barrier to the attainment of equilibrium. It follows that under conditions that would ultimately lead to high surface occupancy at equilibrium, conventional measurements of the kinetics of adsorption may fail to reveal essential aspects of the process occurring on a time scale several orders of magnitude longer than that characterizing the initial low-occupancy (rapid) phase of adsorption. Studies aimed at physico-chemical characterization of poly-amido-amine (PAMAM) dendritic polymers in aqueous solution were initiated. These highly compact, homogeneous polymers may be synthesized to approximately the same size and shape as globular proteins, and their surface residues may be modified (for example, hydroxylated) to minimize reactivity with biopolymers. Such polymers may thus serve as models for the volume-excluding properties of proteins in highly concentrated solution. Measurements have been made of the absorbance spectrum, density increment, refractive increment, sedimentation coefficient, diffusion coefficient, buoyant molar mass (direct measurement) and actual molar mass (indirect measurement). Purity and homogeneity of the polymer were monitored via high-performance size exclusion chromatography. Measurements of intermolecular interaction via sedimentation equilibrium are in progress. A spectrophotometric absorbance scanner based has been designed and built, the purpose of which is to acquire UV-visible absorbance data as a function of both wavelength and radial position from solutions of one or more macromolecular solutes that have been centrifuged to sedimentation equilibrium. The instrument is currently being tested and the design undergoing modification as necessary. Preliminary results suggest the instrument will be capable of providing data of substantially higher spectrophotometric accuracy, precision and spatial resolution than attainable via currently available instrumentation. The technique of tracer sedimentation equilibrium developed earlier in our laboratory has been extended both theoretically and experimentally to permit the thermodynamic activity and/or state of association of a trace amount of labelled macrosolute to be characterized in the presence of an arbitrary concentration of a second macrosolute. Using this technique the thermodynamic activity of dilute fibrinogen (Fbg) has been measured in solutions of bovine serum albumin (BSA) at concentrations of up to 100 mg/ml. It was found that at a BSA concentration of 80 mg/ml, which is comparable to the total concentration of protein in blood serum, the thermodynamic activity of Fbg is ten-fold greater than its concentration, suggesting that the affinity of Fbg for platelet receptors in blood serum may be substantially larger than suggested by binding experiments carried out in vitro. The presence of a substantial population of soluble oligomers of Fbg has been demonstrated in the presence of Ca++ and moderate concentrations (<40 mg/ml) of BSA.
NIH ExPorter
NIH ExPorter