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

Paper ID Volume ID Publish Year Pages File Format Full-Text
35146 45078 2012 7 PDF Available
Title
Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds
Abstract

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 (Ki) 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 °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.

► 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.

Keywords
Entada acaciifolia; Circular dichroism; Kunitz-type inhibitor; Trypsin inhibitor
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Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 47, Issue 6, June 2012, Pages 929–935
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
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Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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