EFFECT OF N-B TRANSITION ON THE MICROENVIRONMENT SURROUNDING (34)CYS IN HUMAN SERUM-ALBUMIN

The effect of pH on the microenvironment surrounding (34)Cys in human serum albumin (HSA) has been studied using acrylodan, a Cys-specific f luorescence probe. The reactivity of (34)Cys with 5,5'-dithiobis(2-nit ro benzoic acid) (DTNB) followed a pseudo-first-order reaction, and th e increase in reactivity mas dependent on pH and oleate content, Compa red with the N-form of HSA-acrylodan conjugate (pH 6.2), the B-form (p H 8.4) has a blue-shifted Em(max) and enhanced fluorescence intensity derived from acrylodan covalently attached to (34)Cys suggesting that the exposure around (34)Cys in the E-form was less than that in the N- form. The conformational change induced bg fatty acid increased tile e xposure around (34)Cys, while that induced by an increase in pH decrea sed it. Further, since tile effect of oleate on the fluorescence of ac rylodan was nearly the same for both conformers, the effects of pH and oleate on the microenvironment surrounding (34)Cys should be independ ent and additive. We concluded that the increase of reactivity of (34) Cys as a function in increasing pH may well be related to an increase in mercaptide ion content.