The interaction of the fluorescent probe 1-anilinonaphthalene-8-sulfonate with Carlsberg subtilisin.

1-Anilinonaphthalene-8-sulfonate (Ans) binds to Carlsberg subtilisin [EC 3.4.21.14] with a large enhancement of its fluorescence intensity and a shift of the emission maximum to shorter wavelength. The present study indicated that one molecule of Ans binds to Carlsberg subtilisin and inhibits the hydrolysis of substrates in a noncompetitive manner. The dissociation constants of Ans-Carlsberg subtilisin complex were 6.5 x 10(-4) M at pH 6.5 and 7.8, respectively, in terms of fluorescence titration, being in accord with the values 5 approximately 8 x 10(-4) M at pH 7.8) obtained from kinetic studies using various substrates. The dissociation constant of N alpha-acetyl-2-(2-nitro-4-carboxyphenylsulfenyl)-L-tryptophan methyl ester (Ac-Trp(NCps)-OMe), which is a competitive inhibitor of the enzyme, however, became 3.3 times greater in the presence of Ans. It was also observed that the fluorescence intensity of the Ans-enzyme complex decreased in the presence of Ac-Trp(NCps)-OMe or N alpha-acetyl-O-trans-p-phenylazobenzoyl-L-tyrosine methyl ester (Ac-Tyr(PABz)-OMe). These phenomena suggest that the Ans binding site is in the vicinity of the active site of the enzyme.