In situ Raman spectroscopy distinguishes between reversible and irreversible thiol modifications in L-cysteine

In this study we introduce in situ Raman spectroscopy as an effective tool to distinguish between reversible and irreversible thiol modifications in L-cysteine, the most critical host for sulphydryls in proteins and enzymes. We place special emphasis on the conditions under which the formation and breakage of disulphide bonds is reversible and produces free thiol groups. Thiol groups from L-cysteine are highly reactive and are frequently converted into disulphide bonds via reaction with mercaptans, including S-methyl methanethiosulfonate (MMTS). It was previously claimed that disulphide groups can be reversibly introduced in and eliminated from L-cysteine in order to restore a free thiol group, using a reductant such as tris(2-carboxyethyl) phosphine (TCEP). Raman spectroscopy is found to effectively monitor the formation and subsequent breaking of disulphide bonds, and demonstrates that although TCEP is effective in breaking disulphide bonds, an excess of TCEP is required to reversibly form free thiol groups from L-cysteine. These results will be critical for cysteine-metal bonding investigations. Our Raman mode assignment in MMTS and TCEP also provides a benchmark for future studies using these compounds.