The purification and crystallization of beef erythrocyte catalase

Abstract

A procedure for the purification and crystallization of catalase from beef erythrocytes with an overall yield of about 25% just before the crystallization step has been developed, which involves adsorption on alumina gel, ethanol precipitation with subsequent CHCl 3 extraction, ammonium sulfate precipitation, and crystallization by dialysis. The molecular weight of this enzyme, as calculated from ultracentrifugation studies, is between 257,000 and 269,000. Spectral analysis show that beef erythrocyte catalase is only slightly reducible by dithionite. The spectrum of the erythrocyte catalase in the visible and near ultraviolet regions is similar in form to that of catalase isolated from beef liver. Beef erythrocyte catalase, however, contains twice the amount of hematin as does beef liver catalase, and the best preparations of erythrocyte catalase are nearly twice as active as beef liver catalase. Amino acid analyses of beef erythrocyte and beef liver catalases do not show differences between the two enzymes with respect to amino acid percentages, so that the most marked dissimilarity is the difference in hematin content. (Small differences in amino acid sequences would not have been detected). The observations on hematin content indicate that all of the hematins of beef erythrocyte catalase contribute to catalytic activity.