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Kunitz-Type Trypsin Inhibitor with High Stability from Spinacia oleracea L. Seeds

Zhuang Kang1, Jia-hong Jiang1, Dong Wang1, Ke Liu1, and Lin-fang Du1,2*

1Key Laboratory of Bio-resources and Eco-environment of the Ministry of Education, Sichuan University, Chengdu 610064, P. R. China

2Institute for Nanobiomedical Technology and Membrane Biology, Sichuan University, Chengdu 610064, China; fax: +86-28-8541-5300; E-mail: dulinfang@yahoo.com

* To whom correspondence should be addressed.

Received September 25, 2007; Revision received February 26, 2008
The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI–trypsin complex (Ki = 2.3·10–6 M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors.
KEY WORDS: Spinacia oleracea L., trypsin inhibitor, protein stability, Kunitz family, Pieris rapae

DOI: 10.1134/S0006297909010167