R. Singh, G. Lamoureux, W. Lees
1995
Citations
3
Influential Citations
38
Citations
Journal
Methods in enzymology
Abstract
Publisher Summary This chapter describes the reagents that reduce disulfide bonds, under nondenaturing conditions at pH 7, faster than does dithiothreitol (DTT) by a factor of ∼5–7. Dithiothreitol and 2-mercaptoethanol (ME) are the most commonly used reagents for the reduction of disulfide bonds. However, both DTT and ME have drawbacks in their use: DTT is slow in reducing disulfide bonds at pH 7; ME is foul smelling and may not completely reduce disulfide bonds owing to its low reduction potential. A new class of dithiol reagents for rapid reduction of disulfide bonds at pH 7 to 8 is based on two requirements: (1) a low p K a value (∼7–8) for their thiol groups, so that a significant proportion of the thiol is in the reactive thiolate form and (2) a high value for their reduction potential. These dithiol reagents reduce disulfide bonds by the mechanism of thiol–disulfide interchange. The chapter describes three reagents—bis(2-mercaptoethyl) sulfone (BMS), meso -2,5-dimercapto- N,N,N',N' -tetramethyladipamide (DTA), and N,N' -dimethyl- N,N' -bis(mercaptoacetyl)hydrazine (DMH)—that have the desirable properties of high reduction potential and p K a values of ∼7.8. These dithiol reagents (BMS, DTA, and DMH) reduce disulfide bonds in proteins under nondenaturing conditions at pH 7 faster than dithiothreitol. All three reagents are conveniently synthesized from readily available materials.