Publisher Summary This chapter discusses the chemical reduction of disulfides. Disulfides are easily and specifically reduced by thiols, which are the most used reagents for this purpose. However, the excess of the thiol used as a reductant has to be removed before it is possible to assay the newly generated SH groups. The methods discussed for the reduction of disulfides by thiols are dithiothreitol (DTT) and dithioerythritol (DTE); and 2-mercaptoethanol. DTT and DTE have also been extensively used to reduce soluble nonprotein disulfides such as cystine and mixed homocysteine-cysteine disulfides. Mercaptoethanol, the simplest water-soluble organic thiol, is “the poor man's DTT,”—that is, mole for mole it is 150-175 times less expensive. Mercaptoethanol is much used especially in conjunction with SDS-gel electrophoresis. Direct chemical reduction is achieved with sodium borohydride or sodium amalgam whereas more specialized reagents include the phosphines and phosphorothioates. For cleavage generating half thiol only, there is sulfitolysis and cyanolysis. Less well controllable and defined is alkaline hydrolysis.

Effect of heating proteins in a vacuum on their assay by the Lowry and biuret methods

Citations

Chemical reduction of disulfides.

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