Red cell hydrolases : II. Proteinase activities in human erythrocyte plasma membranes

A 0.1% Triton X-100 extract of human erythrocyte plasma membranes contained high proteolytic activity as determined by a very sensitive assay utilizing(3)H-acetylated hemoglobin (162 cpm/pmole) as a substrate. Two proteolytic enzymes having optimum activity at pH 3.4 and pH 7.4 were isolated from Sephadex G-100. The protease active at pH 3.4 was 75 times as active as the pH 7.4 enzyme and it was purified 182-fold over the original homogenate and characterized. A linear relationship for activity versus time and activity versus concentration of enzyme was found. The optimum temperature was 37°C and theK m was 1×10(-5) M hemoglobin. No enzyme activation was observed with any cation studied and EDTA had no inhibitory effect; (10MM Fe(+3) and Hg(+2) were inhibitory). The pH 3.4 protease was stable indefinitely at -20°C in 0.1% Triton X-100. Gel electrophoresis was performed on a sodium dodecylsulfate-mercaptoethanol enzyme preparation and two protein bands (mol. wt. 33,000 and 54,000) were evident for the Sephadex G-200 eluate containing the pH 3.4 protease.