S-Nitrosated of cysteine beta-93 in hemoglobin (SNO-Hb) represents a novel derivative of this respiratory protein that has been detected in vivo, but whose biochemical characteristics have yet be fully defined. A mechanism was proposed by which SNO-Hb, via oxygen-sensitive transnitrosation reactions, releases nitric oxide and thus modulates blood flow. However, as yet little is known concerning the effect of S-nitrosation on the oxygen binding characteristics of Hb, or the effects of oxygen on transnitrosation reactions of the beta93Cys reside. These are important issues as they provide a biochemical framework to assess the physiological function of this protein. Our results demonstrate that S-nitrosation of normal human Hb results in a considerable left shift of its oxygen dissociation curve and that this increase in oxygen affinity of SNO-Hb was proportional to the degree of S-nitrosation of Hb. Furthermore, S-nitrosation had no effect on the co-operative nature o f ox ygen binding to hemoglobin, nor did it significantly alter the effects of allosteric regulatory mechanisms of pH (the bohr effect), temperature or inorganic phosphates on oxygen affinity. We further explored the biochemical properties of SNO-Hb with regards to transnitrosation reactions between this protein and GSH. Analysis of this process indicate that it is a second order reversible reaction, that is only slightly sensitive to the oxygenation state of Hb. Simulation of the reaction between SNO-Hb and GSH under physiological conditions suggest, however, that although vasoactive concentrations of GSNO may be formed over approximately 15 secs, this is too slow to account for the currently proposed biological effects of SNO-Hb as a vasodilator.