Reactivity of Sulfenic Acid in Human Serum Albumin

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 (HSA−SO₂H), as detected by mass spectrometry. The rate constants of HSA−SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA−SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA−SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 ± 0.2, 2.9 ± 0.5, 9.3 ± 0.9, and 55 ± 3 M^-1 s^-1 (25 °C, pH 7.4), respectively, supporting a role for HSA−SOH in the formation of mixed disulfides.