A method to study kinetics of transnitrosation with nitrosoglutathione: reactions with hemoglobin and other thiols.
The rate of protein S-nitrosylation, a reversible process by which S-nitroso thiol (RS-NO) compounds exchange the NO+ moiety with protein SH groups, is essentially governed by two factors, the pK alpha and the accessibility of the protein sulfhydryl. A useful method of following transnitrosation kinetics of various protein and nonprotein SH compounds with GS-NO is described. When the reaction is carried out in the presence of 1-chloro-2,4-dinitrobenzene and glutathione transferases, the rate of RS-NO formation (RSH + GS-NO-->RS-NO + GSH) can be monitored by spectrophotometry at 340 nm in terms of the enzymatic conversion of GSH to a GS conjugate. Unlike methods based on NO release from the S-NO bond, this procedure is rapid and accurate and requires relatively small amounts of thiols. The second order rate constants of S-nitrosylation of human and rat oxy- and deoxyhemoglobin of BSA and other thiols were calculated by this method which confirmed previous results reported in the literature.
Rossi
R
R
Department of Environmental Biology, University of Siena, Italy.
Lusini
L
L
Giannerini
F
F
Giustarini
D
D
Lungarella
G
G
Di Simplicio
P
P
D000818:Animals; D005978:Glutathione / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015538:Nitrosation; D009603:Nitroso Compounds / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013053:Spectrophotometry
D006454:Hemoglobins; D009603:Nitroso Compounds; D005982:Glutathione Transferase; D005978:Glutathione