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

• Published in: Process biochemistry (1991) Vol. 47; no. 6; pp. 929 - 935
• Main Authors: de Oliveira, Caio Fernando Ramalho, Vasconcelos, Ilka Maria, Aparicio, Ricardo, Freire, Maria das Graças Machado, Baldasso, Paulo Aparecido, Marangoni, Sergio, Macedo, Maria Lígia Rodrigues
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.06.2012
• Subjects: active sites; amino acid composition; cattle; Circular dichroism; denaturation; dissociation; disulfide bonds; Entada; Entada acaciifolia; heat; Kunitz-type inhibitor; Kunitz-type proteinase inhibitor; molecular weight; polyacrylamide gel electrophoresis; reversed-phase high performance liquid chromatography; seeds; stoichiometry; trypsin; Trypsin inhibitor; urea; Kunitz-type inhibitor; Trypsin inhibitor; Entada acaciifolia; Circular dichroism
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2012.02.022

► EATI is a competitive inhibitor homologous to Kunitz-type inhibitors. ► DTT treatment at high concentration does not alter the inhibitory activity of EATI. ► The reactive site of EATI is not stabilized by disulfide bonds. A new trypsin inhibitor (EATI) was isolated from Entada acaciifolia (Benth.) seeds. EATI is a competitive inhibitor with a molecular mass of 20kDa and an inhibition stoichiometry of 1:1 for bovine trypsin. The dissociation constant (Ki) calculated was 1.75nmol/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°C), pH (2–10), urea (8mol/L) and its inhibitory activity was unaltered in different concentrations of DTT (up to 100mmol/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.