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J. Biochem, 1980, Vol. 87, No. 4 1235-1241
© 1980 Japanese Biochemical Society

research-article

The Interaction of the Fluorescent Probe 1-Anilinonaphthalene-8-Sulfonate with Carlsberg Subtilisin

Yuji KARASAKI and Motonori OHNO

Laboratory of Enzyme Chemistry, Faculty of Science, Kyushu University Higashi-ku, Fukuoka, Fukuoka 812

1-Anilinonaphthalene-8-sulfonate (Ans) binds to Carlsberg subtilisin [EC 3.4.21.14 [EC] ] 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 6.5×10–4M and 8.5×10–4M at pH 6.5 and 7.8, respectively, in terms of fluorescence titration, being in accord with the values (5~ 8×10–4 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.


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