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

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

Autor Dametto, M. Google Scholar
David, A. P. Google Scholar
Azzolini, S. S. Google Scholar
Campos, ITN Google Scholar
Tanaka, A. M. Google Scholar
Gomes, A. Google Scholar
Andreotti, R. Google Scholar
Tanaka, A. S. Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Inst Butantan
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
Resumo 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 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 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 x 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.
Assunto trypsin-like enzyme
fibrinolytic activity
protein purification
hematophagous
Haemotobia irritans irritans
Idioma Inglês
Data 2000-08-01
Publicado em Journal of Protein Chemistry. New York: Kluwer Academic/plenum Publ, v. 19, n. 6, p. 515-521, 2000.
ISSN 0277-8033 (Sherpa/Romeo, fator de impacto)
Editor Kluwer Academic/plenum Publ
Extensão 515-521
Fonte http://dx.doi.org/10.1023/A:1026557600429
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000166011900011
URI http://repositorio.unifesp.br/handle/11600/26352

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