A thiol-activated digestive proteinase from adults of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Complete activation of proteinase(s) from the digestive midgut of adult Rhodnius prolixus occurs in the presence of thiol activator and EDTA. In the assay procedure developed and reported upon, gut homogenate is added to 1.5 ml 0.1 M potassium phosphate buffer, pH 5.5, containing 1.5 mM dithiothreitol (DTT) and 3 mM EDTA, and is allowed to incubate at 30 °C for 5 min. Fifty microlitres of N-benzoyl- DL-arginine naphthylamine (BANA), 40 mg/ml in dimethyl sulfoxide, is added, and the reaction is stopped after incubation at 30 °C by the addition of 2 ml fast garnet GBC salt colour reagent. Greatest activation occurs with dithiothreitol, followed by cysteine, glutathione, and mercaptoethanol in descending order of activation ability. Complete activation of BANA hydrolysis occurs with 0.4 mM DTT and 1.5 mM EDTA. With the assay, 0.2 gut provides ΔA 520 nm of 0.15 in 30 min. The conditions of the assay suggest that a cathepsin B1-like proteinase is present. The proteinase occurs in the midgut lumen and maximal a...