Analysis of Bound Sulfane Sulfur Formation During Alkaline Treatment of Human Serum Albumin

Human plasma contains trace amounts of bound sulfane sulfur (BS) and hydrogen sulfide ions (HS−), but reported concentrations vary widely depending on the analytical method used. Extremely high values have been obtained in methods that use alkaline treatment. To identify the underlying mechanism, human serum albumin (HSA) was incubated at various pH values, and the formation of BS and HS− was analyzed. Incubating HSA in PBS or buffer at pH 8.0–10.0 resulted in no significant changes, whereas BS formed rapidly under strongly alkaline conditions, followed by the generation of free HS− and a continuous increase in their concentrations. In contrast, when the number of thiol (–SH) groups derived from cysteine residues involved in intramolecular disulfide bonds in HSA was quantified after complete reduction, it decreased significantly by an amount corresponding to the total amount of BS and HS− formed. This indicates that the C–S bonds of the cysteine residues that originally formed intramolecular disulfides in HSA were cleaved by β-elimination, resulting in the formation of cysteine persulfide groups. Moreover, SDS–PAGE and SEC analyses revealed obvious structural changes which were indicative of fragmentation and degradation. Further, LC–MS/MS analysis revealed that some cysteine residues were converted to dehydroalanine (Dha) residues. These results suggest that the millimolar levels of polysulfide detected in plasma after alkaline treatment are likely artificial products resulting from the alkaline-induced degradation of intramolecular disulfide bonds within HSA. Therefore, strongly alkaline conditions should be avoided when analyzing BS and HS− in biological samples containing disulfide bonds.