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

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• 作者：Caio Fernando Ramalho de Oliveira^a ; ^e ; Ilka Maria Vasconcelos^b ; Ricardo Aparicio^c ; Maria das Graç ; as Machado Freire^d ; Paulo Aparecido Baldasso^a ; Sergio Marangoni^a ; Maria Lí ; gia Rodrigues Macedo^e ; ^{bioplant@terra.com.br}
• 关键词：Entada acaciifolia ; Circular dichroism ; Kunitz-type inhibitor ; Trypsin inhibitor
• 刊名：Process Biochemistry
• 出版年：2012
• 期刊代码：29_13595113
• 类别：bio
• 出版时间：June, 2012
• 卷：47
• 期：6
• 页码：929-935
• 文件大小：677 K

摘要

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 掳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.