Fluorescence quenching as an indicator for the exposure of tryptophyl residues in Streptomyces subtilisin inhibitor.

Fluorescence quenching by acrylamide and trichloroethanol of two equivalent tryptophyl residues in Streptomyces Subtilisin Inhibitor (SSI) was studied in several different conformations of the protein. Fluorescence intensity and polarization degree were simultaneously observed, and the relation between fluorescence intensity and lifetime was examined. The quenching profiles showed deviation from the Stern-Volmer plots. In the denatured form, a nonstoichiometric mechanism which differs from the static and dynamic one was found to be applicable to the interpretation of quenching by acrylamide. Quenching by trichloroethanol proceeds via the nonstoichiometric process for both the native (N) and the denatured (D) form. Each quenching constant obtained from SSI in N- or D-form for acrylamide or trichloroethanol quenching was compared with the others or with that from free tryptophan, as an indicator of the relative exposure of tryptophyl residues. The quenching constant of acrylamide for the urea denatured D-form is larger than that for the acid denatured D-form. The urea denatured state has more exposed tryptophyl residues. Temperature dependence of fluorescence quenching by Cs+ was also studied and a structural fluctuation was found around Trp-86 prior to the massive unfolding of the hydrophobic core containing the residue.