Effects of temperature, oxygen, heme ligands and sulfhydryl alkylation on the reactions of nitroprusside and nitroglycerin with hemoglobin.

Nitrovasodilators react with hemoglobin (Hb) to form heme(III) and nitric oxide (NO)Hb. These reactions can be exploited as models for events that take place at the cellular level leading to the biological effects of the prodrugs. Sodium nitroprusside (SNP) is known to undergo a one-electron reduction in its reaction with heme(II), resulting in the labilization of the cyanide ligand trans to the NO ligand. This reduced form is here called "penta." Upon dissociation of the trans-cyanide, the resulting species is here called "tetra." Dissociation of the trans-cyanide is obligatory for transfer of the NO to a heme(II) group. NO release from penta is blocked by excess free cyanide in solution, which prevents the formation of tetra. As reported here, both penta and tetra had unique EPR signals when frozen at -196 degrees, but only tetra gave an EPR signal at 22 degrees. NOHb also has a unique EPR signal, but it could not be detected when SNP was incubated with Hb in air or 10 or 5% oxygen. NOHb was detected in similar incubations under 1% oxygen, but the levels were 3- to 10-fold lower than those found under 100% nitrogen. The concentration of tetra was also much lower under 1% oxygen and penta was not detectable, suggesting that oxygen may either shift the penta-tetra equilibrium towards tetra or that penta may be susceptible to oxidation by molecular oxygen. Nitroglycerin (GTN) also generated much less NOHb but more heme(III) under 1% oxygen than under nitrogen. Carbon monoxide (CO), which binds to heme(II), completely blocked the reactions of SNP and GTN with Hb, whereas N-ethylmaleimide (NEM) alkylation of globin sulfhydryl groups increased both NOHb and heme(III) formation. 13C NMR studies on uniformly 13C-labeled SNP suggested that oxygen had little effect on the concentrations of the NMR-detectable species in the reaction. In summary, the most oxygen-sensitive step in the nitrosylation of Hb by SNP was probably the transfer of NO to heme(II). However, the penta-tetra equilibrium was affected by oxygen, temperature and cyanide. No evidence was found for the involvement of the globin sulfhydryl groups in either the GTN or the SNP reaction with Hb.