Some problems in enzyme determination in red cells mainly due to the membrane structure

For the enzyme assay in red blood cells, three problems related to membrane structure were examined. (1) It was difficult to get full activity of membrane enzymes, especially ATPases. The effects of various buffers and substrates and conditions of preservation were examined. (2) During preservation of blood, phosphofructokinase (PFK) decreased most among 15 enzymes including all glycolytic enzymes. This decrease was prevented by the addition of adenine and inosine which produce ATP inside the cells. After hemolysis, PFK decreased most rapidly, too. ATP prevented the inactivation effectively. (3) after centrifugation, even according to Murphy's method, pyruvate kinase decreased most in the lowest layer of stacked erythrocytes. This might be due to the effect of centrifugation.