Authors: Wang, Run; Bright, Frank V.

Source: Applied Spectroscopy, Volume 47, Issue 6, Pages 677-859 (June 1993) , pp. 792-799(8)

Publisher: Society for Applied Spectroscopy

Abstract:

The complexation of 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to the hydrophobic sites of bovine serum albumin (BSA) and the effects of quencher and denaturant were studied by steady-state and dynamic fluorescence spectroscopy. Experimental results show that there are multiple sites in BSA which bind TNS. Fluorescence decay kinetics of BSA-bound TNS were recovered by multifrequency phase-modulation measurements and analyzed by a global analysis scheme. For all the cases, a distributed lifetime model best fits the experimental data—a result of the dynamical nature of BSA/TNS association and of the multiple binding sites in BSA. In addition to the distributed species, a short, discrete component exists, arising from unbound or free TNS. The recovered Stern-Volmer quenching constant for acrylamide from the lifetime data is consistent with the steady-state results. The denaturant, urea, decreases significantly the pre-exponential factor for protein-bound TNS, but affects the center lifetime and the distribution width only at high urea concentrations. The perturbation of probe molecules on the three-dimensional conformation of proteins and the effects of complex (TNS-BSA) age were also studied.

Keywords: Continuous lifetime distributions; Protein/ligand binding; Quencher effects

Document Type: Research article

DOI: http://dx.doi.org/10.1366/0003702934066884

Affiliations: 1: Department of Chemistry, Acheson Hall, State University of New York at Buffalo, Buffalo, New York 14214

Publication date: 1993-06-01

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