NONCOVALENT INTERMOLECULAR INTERACTIONS IN BIOCHEMISTRY

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Statistical thermodynamic and kinetic models have been developed to describe rates and equilibria of reversible adsorption of proteins to surfaces under conditions such that molecules of adsorbed protein may interact with each other via short range steric repulsion and nonspecific attraction leading to the formation of adsorbate clusters. It is found that if attractive forces are sufficiently strong, they may counteract the highly negatively cooperative effect of steric repulsion, leading to equilibrium adsorption isotherms and adsorption progress curves that are as steep as or steeper than those calculated from simple Langmuir (non-cooperative) adsorption theory, in agreement with a variety of experimental observations. The results obtained are qualitatively independent of the assumed shape of the clusters, providing that clusters are represented as convex bodies. The dispersity, size, shape, and self-interaction of generation 5 polyamidoamine dendrimeric polymers with amino- and hydroxyl terminal groups (surfaces) were characterized using several physicochemical techniques. It was found that amino-surface dendrimers form oligomeric aggregates in aqueous solution, even in the presence of high salt concentrations (0.6 M sodium phosphate). In contrast, the hydroxyl- surface polymer G5-OH behaves as a single homogeneous (or paucidisperse) species at low concentration. Measurements of density increment and the sedimentation and diffusion coefficients of G5-OH suggest a more swollen, porous structure than a globular protein of comparable mass. Measurements of the concentration dependence of sedimentation equilibrium of G5-OH in pH 7.2 phosphate buffer indicate the presence of significant electrostatic repulsion overlaid on weakly attractive interactions, leading to the formation of nonspecific aggregates at sufficiently high dendrimer concentration. The spectrophotometric absorbance scanner reported as under construction in last years annual report has been completed and is currently undergoing testing and fine-tuning to achieve the anticipated accuracy and precision.The technique of nonideal tracer sedimentation equilibrium recently developed in our laboratory has been used to measure the effect of high concentrations of an inert polymer, dextran, on the state of association of calf brain tubulin under nonassembly conditions (i.e., under conditions in which large arrays of tubulin, such as microtubules, are not formed). It was found that with increasing dextran concentration, the weight-average molar mass of tubulin increases progressively to several times that of the monomer, indicating the presence of soluble tubulin oligomers which are believed to constitute precursors to the formation of large insoluble arrays. The self-association of the bacterial cell division protein FtsZ has been studied via sedimentation equilibrium. The dependence of the weight-average molar mass on total protein concentration has been measured as a function of the concentration of magnesium ion. Analysis of the data indicates that under the conditions of the experiments, FtsZ forms indefinitely large aggregates with a stepwise equilibrium association constant that decreases slightly with increasing aggregate size, approaching an asymptotic limit. The dependence of the asymptotic limit upon Mg concentration indicates that one Mg ion is taken up for each molecule of FtsZ added to the growing aggregate. - protein adsorption, statistical thermodynamics, dendritic polymers, fiber-optic spectrophotometer, sedimentation equilibrium, protein association