Purificação e caracterização parcial de uma serino-protease tipo tripsina isolada do intestino de larvas de Lutzomyia longipalpis (Diptera, Psychodidae)

Abstract

Visceral Leishmaniasis is a disease of major public health impact. In the New World the diasease is caused by Leishmania infantum chagasi and transmitted mainly by Lutzomyia longipalpis. In adult sandflies, trypsins play an important role during of blood digestion. However, little is known about the action of these proteases in immature forms. In this work we purified and characterized a trypsin-like enzyme from the midgut of L. longipalpis. Enzyme assays performed with benzamidine (a trypsin inhibitor) demonstrated that trypsin is responsible for most of total protein digestion (58%) compared with chymotrypsins (42%). The process of purification by affinity chromatography (p-aminobenzamidine column) produced fractions with tryptic activity (substrate: L-BAPNA), which by SDS-PAGE gel analysis revealed a single band of molecular mass of 20.6 kDa. The benzamidine analysis on SDS-PAGE co-polymerized with gelatin and enzyme assays in the presence of various inhibitors indicated that the enzyme is a trypsin-like protease. Assays performed at different pH showed that the maximum tryptic activity occurs at pH 9.5. The specific activity was 2511.5 U.mg-1 and purification factor of 46.1 times. Kinetic analysis using L-BApNA gave a Km value using L-BApNA of 0.49 mM. Mass spectrometry analysis of the purified material detected a mass of 23311.88 Da (MALDI-TOF). Regarding the effects of Ca2+, Mg2+ and EDTA on the trypsin activity, there was a decrease in tryptic activity only when EDTA was added at concentration 18.75 mM, with no interference by metal ions. This is the first study that reports the purification and characterization of a digestive trypsin on L. longipalpis larvae.