title: Boophilus microplus tick larvae, a rich source of Kunitz type serine proteinase inhibitors.
abstract: Serine proteinase inhibitors from Boophilus microplus tick larvae (BmTIs) were purified by affinity chromatography on a trypsin-Sepharose column. BmTIs presented molecular weight between M(r) 6200 and 18,400 and inhibitory activity for trypsin, HuPK (human plasma kallikrein) and neutrophil elastase. Using ion exchange chromatography, BmTIs were separated in several protein pools named BmTI-A to BmTI-F and BmTI-1 to BmTI-7. All BmTI forms presented inhibitory activity for trypsin with apparent dissociation constants (K(i)) in the nM range. In this work, we describe the purification of BmTI-D, BmTI-2, and BmTI-3. These three inhibitors affected neutrophil elastase and HuPK with K(i) also in nM range. BmTI-D proved to be the best HuPK inhibitor, while BmTI-3 was more efficient for neutrophil elastase with dissociation constants (K(i)) of 12 and 0.5 nM, respectively. BmTI-D, BmTI-2, and BmTI-3 N-terminal amino acid sequences allowed us to include them into the BPTI-Kunitz type serine proteinase inhibitor family. BmTIs purified on trypsin-Sepharose were also used in a bovine immunization assay, resulting in antibody (anti-BmTIs) production.
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil.
authors.forename: Sergio Daishi
authors.initials: SD
authors.lastname: Sasaki
authors.forename: Simone Sant'Anna
authors.initials: SS
authors.lastname: Azzolini
authors.forename: Izaura Yoshico
authors.initials: IY
authors.lastname: Hirata
authors.forename: Renato
authors.initials: R
authors.lastname: Andreotti
authors.forename: Aparecida Sadae
authors.initials: AS
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D018884:Ixodes / Q000737:chemistry*; D007814:Larva / Q000737:chemistry; D019272:Leukocyte Elastase / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D015842:Serine Proteinase Inhibitors; D019272:Leukocyte Elastase