We have developed a simple method for the preparation and purification of a stable valency hybrid of hemoglobin in which the heme groups on the beta subunits are oxidized whereas the alpha subunits are reduced and liganded with nitric oxide. It is prepared in high yield by incubating human red blood cells with glucose, nitrite and methylene blue under anaerobic conditions. The properties of this pigment will be compared with those of the fully reduced, nitrosylated pigment and with those of valency hybrids which have been oxposed to nitric oxide gas. The stabilities of these various nitrosylated forms will be followed under aerobic and anaerobic conditions by isoelectric focusing and visible and electron paramagnetic resonance spectroscopy. Attempts will be made to identify any decomposition products. Mice will be injected with chemicals known to generate methemoglobinemia (nitrite, hydroxylamine, aliphatic nitrites and nitrates, aromatic amino and nitro compounds), and the patterns of appearance and disappearance of the valency hybrids and any nitrosylated species will be followed quantitatively with time. The influence of methylene blue when also injected with these chemicals will be assessed. The oxygen affinity of the valency hybrid species prepared by different methods will be determined as well their rates of oxygenation. The biological activity of various nitrosylated species of hemoglobin will be studied using the inhibition of human blood platelet aggregation and relaxation of rabbit aortic strips as test systems. Experiments have been designed to determine if the nitric oxide occurring naturally in human and animal blood is of endogenous or of exogenous origin and whether or not it is bound to hemoglobin or to valency hybrids. Reaction mixtures which result in the formation of symmetrical valency hybrid species of hemoglobin may also have as many as eight other minor components. Experiments are planned to distinguish whether these are unsymmetrical valency hybrids or glycolsylated valency hybrid species. Test systems are described to detect the conversion of chemicals that relax vascular smooth muscle (nitrite, nitroprusside, azide, hydroxylamine, nitroglycerin, etc.) to nitric oxide, the putative common, active metabolite. The biological activity of various nitrosylated species of hemoglobin as prepared in human red cells will be studied with respect to platelet aggregation and relaxation of vascular smooth muscle.
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