ROLE OF THIOLS IN THE TARGETING OF S-NITROSO THIOLS TO RED-BLOOD-CELLS

We compared the nitric oxide ((NO)-N-.)-releasing characteristics of two NO donors, the S-nitroso adduct of bovine serum albumin (BSANO) and the S-nitroso adduct of L-glutathione (GSNO). In oxygenated phosphate buffer (pH 7.4) and in hemoglobin solution, both NO donors released (NO)-N-. only in the presence of a low molecular weight thiol (the most active was L-cysteine). The requirement of thiol to release (NO)-N-. strongly suggests that a transnitrosation reaction occurs between the S-nitroso adduct of the NO donor and the sulfhydryl group of the NO acceptor. The reaction produced a labile S-nitroso-L-cysteine intermediate that released (NO)-N-.. As shown by spin-trapping experiments, the transnitrosation reaction involved the formation of (NO)-N-. (trapped by 2-(4-carboxyphenyl)-4,4,5,5-tetramethyline-1-oxyl 3-oxide) and S-. radicals (trapped by 5,5'-dimethyl-1-pyrroline N-oxide) of both the NO donors and the NO acceptor (L-cysteine). The reaction leading to S-. radical formation was distinct from the transnitrosation reaction, since it was oxygen-dependent. We suggest that S-. radicals are formed from oxidizing species produced after a reaction between (NO)-N-. and molecular oxygen ((NO2)-N-. is a likely candidate). As for pure (NO)-N-. gas, the major oxidation product of NO donors, in phosphate buffer (pH 7.4), was NO2-, with no formation of NO3-. In the presence of oxyhemoglobin, both NO donors produced only NO3-. BSANO and GSNO showed distinct patterns of (NO)-N-. release both in phosphate buffer and in the presence of hemoglobin. In contrast to BSANO, GSNO oxidized HbO(2) in intact cells at a much slower kinetic rate than with cell lysate or purified hemoglobin. The fast kinetics of BSANO with intact cells suggests binding to the cell surface, where L-cysteine can allow the transport of (NO)-N-. to the cytoplasm. On account of their ability to oxidize (NO)-N-. to NO3-, red blood cells probably represent the last step in (NO)-N-. biotransformation or inactivation. The methemoglobin formed in this process was reduced by the NADH-dependent methemoglobin reductase pathway. Our data suggest that sulfhydryl groups, and especially L-cysteine, play a regulatory role in (NO)-N-. targeting to the red blood cells in plasma, thus buffering the concentration of (NO)-N-.. Moreover, the S-nitroso thiol group of serum albumin may intermediate between cells in the metabolism or bioactivity of (NO)-N-..