Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd |
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Authors: | Macedo Maria Lígia Rodrigues Garcia Viviane Alves Freire Maria das Graças M Richardson Michael |
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Affiliation: | a Laboratório de Purificação de Proteínas e suas Funções Biológicas, Departamento de Ciências Naturais, CPTL, Universidade Federal de Mato Grosso do Sul, CP 210, CEP 79603-011, Três Lagoas, MS, Brazil b Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil c Laboratório de Química e Biomoléculas, Centro de Pesquisa, Institutos Superiores do CENSA (ISECENSA), RJ, Brazil d Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil |
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Abstract: | Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT. |
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Keywords: | Inga laurina Mimosoideae Leguminosae Kunitz inhibitor Primary structure |
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