This proposal is a continuation of our research in understanding the adsorption behavior of proteins and other biopolymers on chromatographic surfaces. Three phases of research are proposed. (1) First, we shall continue our work on the study of the behavior of proteins on surfaces using intrinsic fluorescence spectroscopy. We have already been able to determine the kinetic mechanism of unfolding of alpha-lactalbumin on weakly hydrophobic surfaces. We proposed to examine with this species and other globular proteins the role of various parameters on the thermodynamics of interaction with tailor-made surfaces and the kinetics of the unfolding processes. These parameters will include mobile phase additives such as metal ions, structure of the adsorbent surface, protein surface concentration and nonporous vs. porous beads. (2) We propose to purchase a scanning lifetime fluorescence spectrometer in order to probe more deeply protein conformational changes on adsorbent surfaces. Through lifetime measurements and the dynamics of anistropic decay, we will explore the environment around individual fluorophores as a function of contact time with the surface. Such measurements will allow probing different portions of the protein as structural change occurs. (3) In collaboration with Genentech, we propose to explore the chromatographic and intrinsic fluorescence surface behavior of recombinant growth hormone and a large number of variants. Both point mutations and mutations of sequences of amino acids will be examined. These studies will aid in the understanding of the adsorption and separation mechanism, as well as provide information on the relative stability of individual species.
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