Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds

Autor(es)

Ramalho de Oliveira, Caio Fernando ^{[1, 2]} ; Vasconcelos, Ilka Maria ^[3] ; Aparicio, Ricardo ^[4] ; Machado Freire, Maria das Gracas ^[5] ; Baldasso, Paulo Aparecido ^[1] ; Marangoni, Sergio ^[1] ; Rodrigues Macedo, Maria Ligia ^[2]

Afiliação do(s) autor(es)

^[1] Univ Estadual Campinas, Inst Biol, Dept Biochem, BR-13083970 Campinas, SP - Brazil

^[2] Univ Mato Grosso do Sul, Ctr Biol & Hlth Sci, Dept Food Technol & Publ Hlth, BR-79070900 Campo Grande, MS - Brazil

^[3] Univ Ceara, Dept Biochem & Mol Biol, BR-60451970 Fortaleza, Ceara - Brazil

^[4] Univ Estadual Campinas, Inst Chem, BR-13083970 Campinas, SP - Brazil

^[5] CENSA Higher Inst, Res Ctr, BR-28010970 Campos Dos Goytacazes, RJ - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Process Biochemistry; v. 47, n. 6, p. 929-935, JUN 2012.
• Citações Web of Science: 26

Resumo

A new trypsin inhibitor (EATI) was isolated from Entada acaciifolia (Benth.) seeds. EATI is a competitive inhibitor with a molecular mass of 20 kDa and an inhibition stoichiometry of 1:1 for bovine trypsin. The dissociation constant (K-i) calculated was 1.75 nmol/L, displaying a high affinity between enzyme and inhibitor. Both Native PAGE and RP-HPLC revealed that EATI is composed of four isoinhibitors that share the amino acid composition and the amino-terminal sequence homolog to Kunitz-type inhibitors. EATI is stable to denaturation by heat (up to 70 degrees C), pH (2-10), urea (8 mol/L) and its inhibitory activity was unaltered in different concentrations of DTT (up to 100 mmol/L). CD analysis revealed that EATI in reduced form underwent structural modifications associated with a decrease in thermal and pH stabilities, suggesting that their disulfide bonds are not involved in the structuring of its reactive site, but are important for maintenance of its conformational stability. This behavior makes EATI one of the few inhibitors described in the literature with high DTT resistance. (C) 2012 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 11/09361-0 - Inibidor de tripsina de Entada acaciifolia (EATI): caracterização estrutural e avaliação do potencial inseticida contra a lagarta-do-cartucho (Spodoptera frugiperda)
• Beneficiário: Sergio Marangoni
• Modalidade de apoio: Auxílio à Pesquisa - Regular