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Fig. 6. Chagasin overexpression leads to reduced extracellular CP activity. The secretion products of epimastigotes were obtained by resuspending washed cells in PBS and maintaining them for 3 hours at 28°C. The supernatants were collected by centrifugation at 3000 g and filtered through 0.2 µm membranes. (A) The peptidase activity present in the supernatants were measured in 50 mM Na₂HPO₄, 200 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT, 5% DMSO using 5 µM CBZ-Phe-Arg-AMC. Substrate hydrolysis was fully inhibited by E-64 (not shown). (B) Detection of the cruzipain-chagasin complex in supernatants. Supernatants were diluted in SDS-ßME buffer, not boiled, resolved by SDS-PAGE, blotted onto nitrocellulose and probed with anti-chagasin antiserum. (C) Supernatants were boiled for 20 minutes centrifuged at 10,000 g and the inhibitory activity present in the soluble fraction was determined upon incubation with papain in 50 mM Na₂HPO₄, 100 mM NaCl, 5 mM EDTA, pH 6.5, 2.5 mM DTT for 15 minutes at room temperature. After incubation, the residual activity of papain was measured by the hydrolysis of CBZ-Phe-Arg-AMC. The graphs represent the initial velocities for the substrate hydrolysis curves. The experiments were performed in triplicates and are represented as the mean values with the standard deviations (SD). The analysis of variance was performed using ANOVA and the asterisks indicate the scores that are statistically significant at P<0.001 (A) and P<0.01 (C).

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