Separation of erythrocyte enzymes from hemoglobin by chromatography on Blue-Sepharose.

The spectrophotometric determination of the oxygen consumption of biological samples with Hb02 as oxygen donor and’indicator [ 1,2] has the advantage over conventional methods of being accurate, sensitive and suitable for automation [3,4]. Since interferences due to the erythrocyte enzymes are usually negligible, highly purified hemoglobin preparations are not generally required [5-81. However, when extending the spectrophotometric method to determinations such as quantitation of ethanol or simultaneous measurement of oxygen consumption and glycolysis of isolated cells, the presence of catalase and glycolytic enzymes in the HbOZ preparations became a serious inconvenience. enzymes were products of Boehringer Mannheim (a generous gift of Professor H. F. Schmidt). Sepharose 4B obtained from Pharmacia Uppsala was crosslinked according to [ 131, omitting NaBH4 from the reaction medium. Cibacron blue 3G-A, a product of CibaGeigy Basel, was coupled to the crosslinked Sepharose as in [14].