TY - JOUR
T1 - Formation of Disulfides with Diamide
AU - Kosower, Nechama S.
AU - Kosower, Edward M.
N1 - Funding Information:
The authors aPe grateful for the support of their research on thiols and thiol agents by the National Institutes of Health, the United States-Israel Binational Science Foundation, the Israel Academy of Sciences, the Chief Scientist's Office, Israel Ministry of Health, the European Research Office, the Petroleum Research Fund, the J. S. Guggenheim Foundation, and the National Science Foundation.
Funding Information:
Studies from the authors' laboratory were supported by the National Institutes of Health, Grant AM26912. O.W.G. is an Irma T. Hirschl Career Scientist. We thank Ernest B. Campbell and Michael A. Hayward for excellent technical assistance. 20. W. Griffith, this volume \[63\]. 3 S. Ohmori, H. Kodama, T. Ikegami, S. Mizuhara, T. Oura, G. Isshiki, and 1. Uemura, Physiol. Chem. Phys. 4, 286 (1972).
PY - 1987/1
Y1 - 1987/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0023084124&partnerID=8YFLogxK
U2 - 10.1016/0076-6879(87)43050-4
DO - 10.1016/0076-6879(87)43050-4
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AN - SCOPUS:0023084124
SN - 0076-6879
VL - 143
SP - 264
EP - 270
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -