Red cell hydrolases

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Abstract
A 0.1% Triton X-100 extract of human erythrocyte plasma membranes contained high proteolytic activity as determined by a very sensitive assay utilizing3H-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.