Differential binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid to rhodanese catalytic intermediates.
Studies have been performed to quantitate the binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid (2,8-ANS) to catalytic intermediates of the enzyme rhodanese: the sulfur-substituted form (ES) and the sulfur-free form (E). The molecular 2,8-ANS has not been extensively used for protein studies, and some characterization is presented to demonstrate its usefulness as a probe for apolar binding sites. The molecule 2,8-ANS binds to at least two classes of sites on rhodanese. One class (class 1) is present in the ES form and has a Kd of 1.7 mM. The E form of rhodanese appears to have a second class of sites (class 2) in addition to the class 1 sites. Two independent fluorometric methods of analyzing the class 2 binding of 2,8-ANS to the E form gave an average value for Kd congruent to 179 microM. These fluorometric titrations, together with a Job plot, clearly indicate that 2,8-ANS binds to more than one site on the E form of rhodanese. The apparent apolarity is slightly higher for class 2 sites than for the class 1 sites, but both give Z factors of greater than 85. The substrate thiosulfate is able to displace the probe that is bound to the class 2 sites on the E form of the enzyme. Further, 2,8-ANS is found to be a competitive inhibitor of the catalyzed reaction with an apparent Kd of 170 microM. Circular dichroism measurements detect no significant changes in the average conformation of rhodanese that can be ascribed to the presence of 2,8-ANS.(ABSTRACT TRUNCATED AT 250 WORDS)
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013466:Sulfurtransferases / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase