Biochemical characterization of S-nitrosohemoglobin - Mechanisms underlying synthesis, no release, and biological activity

S-Nitrosohemoglobin (SNO-Hb) has been suggested to act as an endogenous NO donor and physiological regulator of blood pressure. However, the mechanism s responsible for the formation of SNO-Hb and those underlying the release of NO and subsequent biological activity have yet to be elucidated. In the present study, a number of nitrosated oxyhemoglobin (HbO(2)) derivatives ha ve been synthesized and characterized. HbO, can be nitrosated at up to thre e distinct residues, one in the alpha-globin chain and two in the beta-chai n, A beta-chain mononitrosated species (designated "SNO-Hb"), generated by the reaction of HbO(2) and S-nitrosoglutathione, released NO via a thiol-de pendent mechanism involving nucleophilic attack at the nitrosated thiol fun ctionality of SNO-Hb; in the case of glutathione, this process was associat ed with the formation of a mixed disulfide, In contrast, multinitrosated he moglobin species released NO and relaxed vascular smooth muscle by a thiol- independent mechanism. HbO, scavenged potently NO released from SNO-Hb and inhibited its vasorelaxant properties. These data show that the predominant vasoactive species released from SNO-Hb is NO, with HNO a putative interme diate; the presence of a low molecular weight thiol is a prerequisite for t his process. Such observations have important implications for the generati on, metabolic fate, and biological activity of S-nitrosothiols.