Artículos de revistas
Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds
Process Biochemistry. Elsevier, v.47, n.6, p.929-935, 2012
Ramalho de Oliveira, Caio Fernando
Vasconcelos, Ilka Maria
Machado Freire, Maria das Gracas
Baldasso, Paulo Aparecido
Rodrigues Macedo, Maria Ligia
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)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.476929935Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Foundation to Support the Development of Education, Science and Technology of State of Mato Grosso do Sul (FUNDECT - Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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