A Better Disulfide Reducing Agent

Dithiothreitol (DTT) is a common buffer additive in protein biochemistry for breaking disulfide bonds. Among other uses, it keeps proteins unfolded for accurate sizing on electrophoretic gels. Yet DTT is relatively inefficient. Only deprotonated thiol groups can reduce cysteine-cysteine sulfur linkages, but at physiological pH, DTT exists almost exclusively in the protonated form. Now researchers describe a new, and seemingly more effective, alternative (J. Am. Chem. Soc., DOI: 10.1021/ja211931f). the University of Wisconsin, Madison’s Ronald T. Raines and coworkers synthesized dithiobutylamine (DTBA) from a cheap, abundant starting material—aspartate. The two compounds have comparable reducing potentials, but DTBA sports lower pK_a values. As an added bonus DTBA, unlike DTT, is nearly odorless. DTBA reduced oxidized β-mercaptoethanol and glutathione three to five times as fast as DTT did. DTBA also reduced papain, with its relatively anionic active site, 14 times as fast as DTT did; creatine kinase, with its more cationic active site, was reduced equally efficiently by both compounds. DTBA is not yet commercially available, but it can be prepared from l-aspartic acid in six steps.