The partial purification and characterisation of serine protease activity in midgut of larval Helicoverpa armigera

Serine protease activity has been extracted and partially purified from the alimentary tract of larval Helicoverpa armigera. The major activity was present in the midgut contents. Characterisation of this protease, using BApNA as substrate, gave a pH optimum of pH 9.5–10. K_m and V_max were 0.254 ± 0.032 mM and 0.351 ± 0.037 μmol pNA produced min⁻¹ mg protein⁻¹ respectively. Inhibition was effected by TLCK but not TPCK; this together with the failure to hydrolyse BTpNA or SUPHEPA, indicated trypsin-like but not chymotrypsin-like specificity. Comparison between the insect protease and bovine trysin revealed differences in inhibitor sensitivity; the insect protease being unaffected by OMTI, whilst showing greater inhibition by chymostatin and SBTI. The kinetics of the interactions between the insect protease activity and various plant-derived protease inhibitors were determined. Unlike bovine trypsin, the insect enzyme was not affected by calcium ions or the divalent chelating agent, EDTA. Partial purification by ion-exchange chromatography, followed by SDS-PAGE, showed that protease activity was largely associated with a polypeptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{⋍ 24,000}$.