Experiments aimed at assessing the utility of fluorescently labeled proteins and fluorescent protein-binding dyes as tracers in sedimentation equilibrium experiments designed to study heteroassociations in solution were completed. Results showed marked nonlinearity of fluorescence intensity with concentration as well as significant dependence of intensity upon the concentration of unlabeled species present. On the basis of these results further attempts to utilize fluorescent labels as a quantitative measure of relative concentration in analytical ultracentrifugation have been abandoned. The Affinity Biosensors IASYS evanescent wave biosensor was tested in our laboratory as a method for the quantitative measurement of the kinetics and/or equilibria of reactions between mobile macromolecular ligands and surface-immobilized specific acceptor molecules. The instrument was found to suffer from a degree of baseline instability that rendered quantitative measurement of either kinetics or equilibria problematic. Following intensive but ultimately unsuccessful attempts to devise techniques for either alleviating the instability or to develop techniques for working around it, the instrument was returned to the manufacturer with a detailed report of our investigations and findings. A novel flow cell for a centrifuge tube microfractionator, incorporating a fiber-optic spectrophotometer, described in last year's annual report, was thoroughly tested for optical linearity and sensitivity. Contrary to prior expectations, the optical performance of the fiber-optic spectrophotometer did not exceed that currently obtainable with the standard absorbance optics of the Beckman XL-A analytical ultracentrifuge. We are currently considering modifications of the design to improve performance. Statistical thermodynamic models for the equilibrium adsorption of proteins to surfaces have been developed which allow for the possibility of proteins adsorbing in multiple conformations distinguished by differing intrinsic free energies of adsorption and surface """"""""footprints"""""""". Calculations exploring the behavior of such systems under a variety of conditions are in progress. A novel strategy for the simulation and modeling of sedimentation equilibrium in highly nonideal solutions of self-associating and hetero-associating solutes has been developed. Computer programs based on this strategy have been successfully implemented and tested. An extremely rapid and accurate semiempirical analysis of sedimentation data obtained from highly nonideal self-associating systems has been developed, paving the way for the use of sedimentation equilibrium as a practical and simple (as well as uniquely powerful) technique for the study of associations in solutions of high total macromolecular content. New techniques for the analysis of sedimentation equilibrium data obtained from solutions containing two macrosolute components have been developed. By using multiple analytical techniques in a hierarchical fashion, it has been found that as the amount of data of different types that is included in the total set of data to be analyzed globally (in contrast to the amount of data of the same type) increases, the ability of the modeling system to discriminate between alternative candidate schemes for self- and hetero-association, and the numerical accuracy of returned results, improve substantially. A new method for rapid estimation of the molar mass and sedimentation and diffusion coefficients of a solute species, based upon real-time modeling of concentration gradients obtained shortly after commencement of a sedimentation experiment, has been developed. The method and software implementation have been tested on seven different proteins with molar masses ranging between 13 and 250 kg. The molar mass and sedimentation and diffusion coefficients of each protein in this group were evaluated with an accuracy of better than 10% within 30 minutes of the start of each experiment.
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