Abstract
The complexation of 2-(<i>p</i>-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.
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