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.
Keywords: BCN-Bio1; Disulfide; Human serum albumin; Hydrogen peroxide; Sulfenic acid; Sulfenylamide; Sulfinic acid; Sulfonic acid; Thiol.
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