Additional biochemical studies have been carried out to investigate the effects of the presence of an unstable hemoglobin (Hb Sabine) on metabolism of erythrocytes. In vitro incubation under physiological conditions of these erythrocytes for periods of 5-15 hours has been utilized to evaluate effects of various additives on metabolism of the cells. The addition of adenine to the blood stimulates adenine nucleotide biosynthesis and is effective in maintaining levels of adenine nucleotides. An excessive rate of breakdown of adenine nucleotides to hypoxanthine has been shown previously to be a major factor in the inability of these cells to maintain ATP. The addition of azide during in vitro incubation proved detrimental to the metabolism of these abnormal erythrocytes. This finding indicates that the immature erythrocytes of the blood are relying on oxidative phosphorylation to meet a portion of their energy needs. Neither pyruvate nor sucrose provided significant beneficial effects on the metabolism of the cells. Methylene blue stimulated the formation of 2,3-diphosphoglycerate, but proved harmful to the maintenance of adenine nucleotides. Effects of precipitation of denatured globin from hemoglobin Sabine on reduced glutathione and on the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, were also studied following in vitro incubation. The precipitating beta polypeptide chains apparently do form disulfide bridges with reduced glutathione. However, no disulfide bridge formation with glyceraldehyde-3-phosphate dehydrogenase was noted during a 15-hour in vitro incubation.
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