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

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Abstract
Complete activation of proteinase(s) from the digestive midgut of adult R. prolixus occurs in the presence of thiol activator and EDTA. In the assay procedure reported, 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.degree. C for 5 min. BANA (N-benzoyl-DL-arginine naphthylamine, 50 .mu.l, 40 mg/ml in dimethyl sulfoxide, is added, and the reaction is stopped after incubation at 30.degree. C by the addition of 2 ml fast garnet GBC salt color 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 DDT and 1.5 mM EDTA. With the assay, 0.2 gut provides .DELTA.A 520 nm of 0.15 in 30 min. Assay conditions suggest presence of a cathepsin B1-like proteinase. The proteinase occurs in the midgut lumen and maximal activity occurs 5-10 days after ingestion of the blood meal.

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