Leishmania (V) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary
leishmaniasis (cutaneous and mucosal). The drugs of choice used in
leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory
infections. Among the promising new targets for anti-protozoan
chemotherapy are the
proteases. In this study,
serine proteases were partially purified from aqueous,
detergent and extracellular extracts of Leishmania braziliensis promastigotes by
aprotinin-
agarose affinity chromatography. By zymography, the
enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the
detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified
proteases exhibited
esterase activity against Nalpha-
benzoyl-L-arginine ethyl ester hydrochloride and Nalpha-p-tosyl-
L-arginine methyl ester hydrochloride (
serine protease substrates) and optimal activity at pH 8. 0.
Proteases purified from the aqueous and extracellular extracts were effectively inhibited by
benzamidine (
trypsin inhibitor) and those from the
detergent extract were inhibited by N-tosyl-L-phenyl-
alanine chloromethyl ketone (
chymotrypsin inhibitor) indicating that all these
enzymes are
serine proteases. These findings indicate that L. braziliensis
serine proteases display some biochemical similarities with L. amazonensis
serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a
serine protease from Leishmania braziliensis.