Reagents for rapid reduction of disulfide bonds in proteins

Abstract

Publisher Summary Disulfide-reducing reagents are routinely used in biochemical manipulations for (i) reducing the native disulfide bonds in proteins and (ii) maintaining the essential thiol groups in proteins by preventing their oxidation to the disulfide state. Dithiothreitol (DTT) is the most popular disulfide-reducing reagent. This chapter discusses the comparison of reactivities of bis(2-mercaptoethyl)sulfone (BMS), [N,N’-dimethyl-N,N’-bis(mercaptoacetyl)hydrazine (DMH), and DTT toward disulfide groups in several proteins under non-denaturing conditions at pH 7. Both BMS and DMH reduce disulfide bonds in proteins at pH 7 faster than DTT does by a factor of ∼5-7 in non-denaturing conditions. BMS is, therefore, more reducing than DMH and slightly less reducing than DTT. All these dithiols (BMS, DMH, and DTT) have significantly high reduction potentials and reduce noncyclic disulfides completely. Although, both BMS and DMH reduce disulfides at similar rates, the chapter recommends the use of BMS, because it is commercially available, it is odorless, and it has a high reduction potential.