Proteinases larvares de Dermatobia hominis (Linnaeus Jr., 1781) (DIPTERA: CUTEREBRIDAE)

Abstract

We performed a combination of proteinase assay, either in solution or immobilized in sodium dodecyl sulfate-polyacrylamide gel copolymerized with gelatin, to detect and quantify proteinases of Dermatobia hominis second (L2) and third (L3) instar larvae. In quantitative assays, we examined proteinase activity by hydrolysis of a panel of peptide bonds specific for the main proteinase classes. We verified that the pGlu-Phe-Leu p-nitroanilide substrate was hydrolyzed by crude extracts of L2 (3.0 ± 0.2 nmoles hour-1 mg of protein-1) and L3 (7.7 ± 0.1 nmoles hour-1 mg of protein-1) and that both activities were partially inhibited by transepoxysuccinyl- L-leucylamido-(4-guanidino)butane, 15 % and 3 % respectively. Also, we demonstrated that the Na-p-Tosyl-L-Arg methyl ester substrate was hydrolyzed by crude extracts of L2 (117 ± 24 nmoles hour-1 mg of protein-1) and L3 (111 ± 10 nmoles hour-1 mg of protein-1), suggesting a predominance of esterase activity in the crude larval preparation. Interestingly, the specific activity of serine-proteinases was totally inhibited by Phenylmethylsulphonyl fluoride in the L3 crude extract, while only 10 % of this enzyme class activity was inhibited in the L2 crude extract. Also, we have detected crude extract L2 (Km = 7,59) larvae have more affinity than L3 larvae (Km = 35,75) to Na-p-Tosyl-L-Arg methyl ester. The results of the qualitative assays with substrate gels suggested that L2 and L3 larvae express serine-proteinases with similar (13 kDa and 22 kDa) and distinct (50 kDa in L2 and 30 kDa in L3) relative molecular masses. Additionally, we have isolated an enriched esterase activity from L3 crude extract using successive chromatographies in Aprotinine-Agarose and DEAE-Sephacell columns. By this strategy we detected only one 50 kDa proteinase in this larvae crude extract. Finally, these findings contribute to the biochemical characterization of D. hominis L2 and L3 larvae.