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-n itroso thiol (RS-NO) compounds exchange the NO+ moiety with protein SH groups, is essentially governed by two factors, the pK(a) and the acc essibility of the protein sulfhydryl. A useful method of following tra nsnitrosation kinetics of various protein and nonprotein SH compounds with OS-NO is described. When the reaction is carried out in the prese nce of 1-chloro-2,4-dinitrobenzene and glutathione transferases, the r ate 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-nitrosyl ation of human and rat oxy-and deoxyhemoglobin of BSA and other thiols were calculated by this method which confirmed previous results repor ted in the literature. (C) 1997 Academic Press.