Formation of Disulfides with Diamide

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This chapter discusses the formation of disulfides with diamide. Several diazenecarbonyl derivatives were found to be active for the conversion of thiols to disulfides. The most convenient and chemically simple agent was diazenedicarboxylic acid bis(N,N-di-methylamide), now known by the trivial name, “diamide.” Diamide is a yellow, nonhygroscopic solid, easily soluble in both water and organic solvents, and rather stable toward hydrolysis. Diamide penetrates cell membranes within seconds and also reacts within the cell at a high rate (seconds to minutes) at physiological pH. Diamide is effective in the absence of oxygen, conditions under which metabolic activity is minimal. There is a simple stoichiometric relationship between the amount of agent added and the quantity of thiol reacted. Because the reaction of diamide with thiols has a low activation energy, thiol oxidation is fast at low temperatures. Reaction can be terminated through removal of diamide by washing of the cells or stopped instantaneously by the addition of acid. In most cases, diamide treatment does not cause any irreversible damage, and, after incubation of the cells with appropriate substrates at a suitable temperature, the original thiol status is recovered. Diamide treatment thus allows the study of cell functions altered by a perturbation in thiol status.

Original languageEnglish
Pages (from-to)264-270
Number of pages7
JournalMethods in Enzymology
Issue numberC
StatePublished - Jan 1987


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