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PublisherThe University of Arizona.
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AbstractThe perturbation of fluorescence in both bovine and human serum albumin caused by chloride, iodide, acrylamide and N-bromosuccinimide was studied under various experimental conditions. Serum albumin fluorescence lifetime changes induced by pH and added solutes were also studied, both in acid solutions and in powders. In general, the two proteins behave similarly. During the N-F transitions, the fluorescence lifetimes and the fluorescences intensities decrease in the same qualitative manner. Chloride binding enhances the fluorescence intensity, but has little or no effect on the fluorescence lifetimes. Chloride enhances the human serum albumin fluorescence intensity much more than it enhances that of bovine serum albumin. Iodide and acrylamide quench both the fluorescence intensities and lifetimes. Acrylamide quenching is hardly affected by pH changes, but is sensitive to the protein concentration. In acrylamide quenching, acrylamide molecules are partitioned into the protein matrix, causing both dynamic and static quenching. Iodide quenching is sensitive to pH, with a maximum quenching at pH 4.0. Iodide quenching decreases with increased ionic strength and with increased protein concentration. The Stern-Volmer plots obtained with iodide as the quencher are downward curving in both proteins. The downward curvature is a result of iodide binding, the main quenching mechanism. Both tryptophans in bovine serum albumin tryptophans and the single human serum albumin tryptophan are very close to the surface of the protein. The environments of the bovine serum albumin tryptophans are not very different from each other. The fluorescence lifetimes of serum albumin powders separated at pH 6.0 are very sensitive to hydration, while the lifetimes of powders separated at pH 2.0 are not. Acrylamide and iodide quench the fluorescence lifetimes of bovine serum albumin powders, even in the driest samples. Quenching is maximum at a hydration approximately equal to that required for monolayer coverage.