caa a22 4500 475743970 CHVBK 20180406123507.0 cr unu---uuuuu 170329e20000801xx s 000 0 eng 10.1023/A:1026557600429 doi (NATIONALLICENCE)springer-10.1023/A:1026557600429 Purification and Characterization of a Trypsin-Like Enzyme with Fibrinolytic Activity Present in the Abdomen of Horn Fly, Haematobia irritans irritans (Diptera: Muscidae) [Elektronische Daten] [M. Dametto, A. David, S. Azzolini, I. Campos, A. Tanaka, A. Gomes, R. Andreotti, A. Tanaka] This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K m values determined for three different substrates were 1.88 × 10−4, 1.28 × 10−4, and 1.40 × 10−4 M for H-α-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 inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, 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 only weak inhibition, e.g., benzamidine with K i = 3.0 × 10−4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect. Plenum Publishing Corporation, 2000 trypsin-like enzyme nationallicence fibrinolytic activity nationallicence protein purification nationallicence hematophagous nationallicence Haematobia irritans irritans nationallicence Dametto M. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut David A. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut Azzolini S. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut Campos I. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut Tanaka A. Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil aut Gomes A. EMBRAPA-Gado de Corte, Campo Grande, MS, Brazil aut Andreotti R. EMBRAPA-Gado de Corte, Campo Grande, MS, Brazil aut Tanaka A. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/6(2000-08-01), 515-521 0277-8033 19:6<515 2000 19 10930 https://doi.org/10.1023/A:1026557600429 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1023/A:1026557600429 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Dametto M. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- David A. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Azzolini S. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Campos I. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Tanaka A. Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Gomes A. EMBRAPA-Gado de Corte, Campo Grande, MS, Brazil aut NATIONALLICENCE 700 1- Andreotti R. EMBRAPA-Gado de Corte, Campo Grande, MS, Brazil aut NATIONALLICENCE 700 1- Tanaka A. Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, SP, Brazil aut NATIONALLICENCE 773 0- Journal of Protein Chemistry Kluwer Academic Publishers-Plenum Publishers 19/6(2000-08-01), 515-521 0277-8033 19:6<515 2000 19 10930 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer