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The effect of pH on the microenvironment surrounding 34Cys in human serum albumin (HSA) has been studied using acrylodan, a Cys-specific fluorescence probe. The reactivity of 34Cys with 5,5'-dithiobis(2-nitro benzoic acid) (DTNB) followed a pseudo-first-order reaction, and the increase in reactivity was dependent on pH and oleate content. Compared with the N-form of HSA-acrylodan conjugate (pH 6.2), the B-form (pH 8.4) has a blue-shifted Emmax and enhanced fluorescence intensity derived from acrylodan covalently attached to 34Cys suggesting that the exposure around 34Cys in the B-form was less than that in the N-form. The conformational change induced by fatty acid increased the exposure around 34Cys, while that induced by an increase in pH decreased it. Further, since the effect of oleate on the fluorescence of acrylodan was nearly the same for both conformers, the effects of pH and oleate on the microenvironment surrounding 34Cys should be independent and additive. We concluded that the increase of reactivity of 34Cys as a function in increasing pH may well be related to an increase in mercaptide ion content.

Effect of N-B Transition on the Microenvironment Surrounding 34Cys in Human Serum Albumin.