Human serum albumin (HSA) contains 17 disulfides and only one reduced cysteine, Cys34, which can be oxidized to a relatively stable sulfenic acid (HSA-SOH). This derivative has been previously detected and quantified. However, its properties are poorly understood. Herein, HSA-SOH formation from the exposure of HSA to hydrogen peroxide was confirmed using the sulfenic acid probe bicyclo [6.1.0]nonyne-biotin (BCN-Bio1), and by direct detection by whole protein mass spectrometry. The decay pathways of HSA-SOH were studied. HSA-SOH reacted with a thiol leading to the formation of a mixed disulfide. The reaction occurred through a concerted or direct displacement mechanism (SN2) with the thiolate (RS−) as nucleophile towards HSA-SOH. The net charge of the thiolate affected the value of the rate constant. In the presence of hydrogen peroxide, HSA-SOH was further oxidized to sulfinic acid (HSA-SO2H) and sulfonic acid (HSA-SO3H). The rate constants of these reactions were estimated. Lastly, HSA-SOH spontaneously decayed in solution. Mass spectrometry experiments suggested that the decay product is a sulfenylamide (HSA-SN(R′)R″). Chromatofocusing analysis showed that the overoxidation with hydrogen peroxide predominates at alkaline pH whereas the spontaneous decay predominates at acidic pH. The present findings provide insights into the reactivity and fate of the sulfenic acid in albumin, which are also of relevance to numerous sulfenic acid-mediated processes in redox biology and catalysis.

Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the pKa value in delipidated HSA is 8.1±0.2 at 37°C and 0.1M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H2O2 is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous SN2 transition state (TS) with calculated Δ‡G and Δ‡H barriers at 298K of respectively 59 and 54kJmol−1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA–SO− and water as products. The structured reaction site cages H2O2, which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H2O2. The lack of reaction-site features required for differential stabilization of the TS (positive charges, H2O2 HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA–SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA–SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H2O2.

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 approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees 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 degrees 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 degrees 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 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.