Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera : Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irr itans irritans (Diptera: Muscidae). The enzyme was purified using a one-ste p process, consisting of affinity chromatography on SBTI-Sepharose. The pur ified protease showed one major active proteinase band on reverse zymograph y with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with m aximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. T he K-m values determined for three different substrates were 1.88 x 10(-4), 1.28 x 10(-4), and 1.40 x 10(-4) M for H-alpha -benzoyl-Ile-Glu-Gly-Arg-p- nitroanilide (S2222), DL-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip -Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibite d by typical serine proteinase inhibitors such as SBTI (soybean trypsin inh ibitor, K-i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K-i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K-i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively ). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K-i value of 39 pM. Synthetic serine protease inhibitors presented o nly weak inhibition, e.g., benzamidine with K-i = 3.0 x 10(-4) M and phenyl methylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified tr ypsin-like enzyme also digested natural substrates such as fibrinogen and f ibrin net. The protease showed higher activity against fibrinogen and fibri n than did bovine trypsin. These data suggest that the proteolytic enzyme o f H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adap tation resulting from the feeding behavior of this hematophagous insect.