S-nitrosothiols in the blood: roles, amounts, and methods of analysis.

In 1991 to 1992, we reported that both endogenous and exogenous nitric oxide (NO) react with thiols in proteins such as albumin to form long-lived S-nitrosothiols (SNOs) with vasodilatory activity.1 We also described the presence of a circulating pool of S-nitrosoalbumin in plasma whose levels were coupled to NO synthase (NOS) activity. Inhibition of NOS led to a decline in SNO-albumin with concomitant production of low-mass SNOs.2 We proposed that SNO-albumin provides a reservoir of NO bioactivity that might be utilized in states of NO deficiency, and that vasodilation by SNO-albumin is transduced by the smallmass SNOs with which it exists in equilibrium. Shortly thereafter,3 we determined that a key low-mass SNO in biological systems is S-nitrosoglutathione (GSNO); that GSNO, in contrast to NO, retained smooth muscle relaxant activity in the presence of blood hemoglobin; and that GSNO is a more potent relaxant than SNO-proteins. Subsequently, we demonstrated the existence of intraerythrocytic equilibria between NO bound to the thiol of glutathione and reactive thiols (cys 93) of hemoglobin on the one hand,4 and NO bound to thiols of hemoglobin and membrane-associated band 3 protein (AE1), on the other hand.5 The exchange of NO groups between S-nitrosohemoglobin (SNO-Hb) and the red blood cell (RBC) membrane is governed by O2 tension (PO2): RBCs dilate blood vessels at low PO2, and the production of membrane SNO is required for vasodilation. In peripheral tissues, blood flow is determined by variations in hemoglobin O2 saturation that are coupled to metabolic demand. The mechanism through which the O2 content of blood evokes this response and the basis for its impairment in many diseases (including heart failure, diabetes, and shock) have been major and longstanding questions in vascular physiology. Our studies suggested that the answers reside with hemoglobin’s ability to serve as both an O2 sensor and O2-responsive transducer of vasodilator activity (Figure). It would later be determined that albumin and hemoglobin are privileged sites of SNO production. In albumin, both a hydrophobic pocket and bound metals (copper and perhaps heme) can facilitate S-nitrosylation by NO,8,9 whereas hemoglobin has several channels through which it can react with NO,10,11 nitrite,12 or GSNO4,13 to produce SNO-Hb (Figure). Additional studies indicated that S-nitrosylation of blood proteins may be catalyzed by superoxide dismutase (SOD), ceruloplasmin, and nitrite. In particular, ceruloplasmin catalyzes the conversion of NO to GSNO,14 and NO in solution or derived from GSNO is targeted by SOD to cys 93 in hemoglobin (rather than heme iron).11,13 A similar mechanism (involving SOD and nitrite) may operate in albumin. Numerous laboratories have verified the presence of SNOalbumin, GSNO, and SNO-Hb in blood and tissues of both animals and humans. However, the amounts that form, the suitability of various methods for assaying various SNOs, and the physiological roles of these molecules remain controversial.