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Abstract

Pathogenic protozoan proteases play crucial roles in the host–parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin–agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS–PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 °C, respectively, determined using α-N-ρ-tosyl-l-arginyl-methyl ester (l-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 °C and after 24 h of pre-treatment at 37 °C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin β-chain was also hydrolyzed by the protease, and four peptidic bonds (L11–V12, E13–A14, L15–Y16, and Y16–L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.