Met-form hemoglobins in long-term stored ACD blood.

On passing ACD blood stored for a year at 4 degrees C through a column of Sephadex G-75, the surviving erythrocytes were separated from hemolyzed cells and plasma. About 18% of stored erythrocytes survived a year of storage, and were spherocytic. The concentration of total ferric hemoglobins in the surviving erythrocytes was about 75% of that in the supernatant. The met-form hemoglobins in the erythrocytes and hemolytic solutions were further analyzed by isoelectric focusing electrophoresis, and (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 valency hybrids were found in addition to methemoglobin. Since the activity of NADH cytochrome b5 reductase was extremely low in the surviving erkythrocytes, the difference in proportion of ferric hemoglobins (valency hjybrids + methemoglobin) in these two fractions might be due simply to the difference in pH between intracellular and extracellular fluids, which is largely reflected in the oxidation rate of hemoglobin. The course of hemoglobin oxidation seems to be through hemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 to methemoglobin.