PURIFICATION AND SOME PROPERTIES OF AN ACIDIC PROTEASE FROM EPIMASTIGOTES OF TRYPANOSOMA-CRUZI

An acidic protease was purified to electrophoretic homogeneity (34-fold purification, 7% yield) from epimastigotes of T. cruzi, Tulahuen strain, Tul 2 stock. The enzyme is monomeric, with a MW of .apprx. 60,000. The purified enzyme was able to use as substrate azocasein, casein, bovine serum albumin and Hb; the highest activity was found with bovine serum albumin at pH 4.0. Soluble T. cruzi proteins were also used as substrates, with an optimum pH of .apprx. 3.0. The purified enzyme was rather thermostable; 50% of the enzyme activity was lost upon preincubation at 62.degree. C for 10 min at pH 5.0. Accordingly, the optimal temperature for the reaction with azocasein as substrate was 60.degree. C. The enzyme was strongly inhibited by the thiol reagents p-chloromercuribenzoate, phenolphthalein mercuric acetate and p-chloromercuriphenylsulfonate (I50 values of 1.0, 1.2 and 3.0 .times. 10-6 M, respectively); thiol-containing compounds, such as 2-mercaptoethanol and reduced glutathione, activated the enzyme; the former was also able to reactivate the enzyme inhibited by the mercurials. N-.alpha.-p-Tosyl-L-lysine chloromethyl ketone and N-.alpha.-p-tosyl-L-phenylalanine chloromethylketone were also strong inhibitors (I50 of 1.2 .times. 10-6 and 1.7 .times. 10-5 M, respectively); 2-mercaptoethanol did not revert these inhibitions. The properties of the purified protease suggested that it may be the main factor responsible for the proteolysis of endogenous substrates that was described in cell-free extracts of T. cruzi.