Reactivity of Sulfenic Acid in Human Serum Albumin

TLDR: The reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity.

Abstract: Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA−SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was ∼20-fold faster than the second phase and first order in HSA−SOH and TNB (105 ± 11 M-1 s-1, 25 °C, pH 7.4), allowing quantitative data on HSA−SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 °C, 4 min) yielded 0.18 ± 0.02 mol of HSA−SOH per mol of HSA. HSA−SH reacted with hydrogen peroxide at 2.7 ± 0.7 M-1 s-1 (37 °C, pH 7.4), while HSA−SOH reacted at 0.4 ± 0.2 M-1 s-1, yielding sulfinic acid ...