Fig. 4

Inhibition of trypsin and chymotrypsin activity by rLdISP1 and rLdISP2 proteins. Midguts were isolated from sand fly at different time intervals (0 to 72 h) after rabbit blood-feeding. The enzymatic assay was performed to observe the maximum trypsin (a) and chymotrypsin (b) activity. c Inhibition of the sand fly trypsin and chymotrypsin activity was analysed by zymography. The midgut isolated at 36 h ABF (1 gut/lane) was either left untreated or treated with rLdISP1, rLdLdISP2, benzamidine or TPCK and PMSF. Residual trypsin (Lanes 2–6) and chymotrypsin (Lanes 7–11) activities were measured by using specific chromogenic substrate. d-g Inhibition of trypsin and chymotrypsin activity was analysed by enzymatic assay. Bovine trypsin, chymotrypsin and the sand fly midgut extract were either left untreated or treated with rLdISP1 (1–10 μM), rLdISP2 (0.1 μM-1 μM) and ecotin (0.1 μM). The experiments were performed in triplicates, and data are means ± SD from three separate experiments. A Student’s t-test was performed for statistical analysis using GraphPad Prism software v5.0. An asterisk (*) denotes P ≤ 0.05, double asterisks (**) denote P ≤ 0.005, and ‘ns’ denotes non-significant as compared to untreated controls or WT as applicable. Abbreviations: T, trypsin; C, chymotrypsin; E, ecotin; G, midgut extract; S, substrate