title: Identification and Characterization of a Trypsin Inhibitor from Fagopyrum tataricumSeeds.
abstract: This study was aimed at investigating the purification and identification of serine protease inhibitors, F. tataricum trypsin inhibitor (FtTI) from tartary buckwheat (Fagopyrum tataricum) seeds. The FtTI was isolated by anion exchange chromatography, affinity chromatography, and centrifugal ultrafiltration. Under reducing and nonreducing conditions, an SDS-PAGE analysis showed that the isolated protein consists of a single polypeptide chain with a molecular mass of approximately 14 kDa. The two isoforms of FtTI were confirmed by the mass spectrometric profile where the two peaks corresponded to 11.487 and 13.838 kDa. The complete amino acid sequence of FtTI has been established by automatic Edman degradation and mass spectrometry. The molecule of FtTI consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of FtTI contains an Asp66-Arg67 bond. The Ki value was calculated using the equation for slow tight binding inhibition which was 1.6 nM for trypsin. FtTI retained its inhibitory activity over a wide range of pH (3-10) and temperature (20-80 °C). FtTI can be rapidly inactivated by the combination of high temperature and high pressure. An analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor Ι family. Furthermore, FtTI exhibited a strong inhibitory activity against phytopathogenic fungi.
authors.affiliation: College of Life Science, Sichuan Agriculture University, Yaan, 625014, Sichuan, People's Republic of China.
authors.forename: Jing-Jun
authors.initials: JJ
authors.lastname: Ruan
authors.affiliation: Institute of Biology, Sylvius Laboratory, Leiden University, Sylviusweg 72, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.
authors.forename: Mei-Liang
authors.initials: ML
authors.lastname: Zhou
authors.affiliation: College of Life Science, Sichuan Agriculture University, Yaan, 625014, Sichuan, People's Republic of China. chen62hui@yahoo.com.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: College of Life Science, Sichuan Agriculture University, Yaan, 625014, Sichuan, People's Republic of China.
authors.forename: Ji-Rong
authors.initials: JR
authors.lastname: Shao
mesh_terms: D019612:Fagopyrum / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology; D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D013696:Temperature
chemical_list: D014361:Trypsin Inhibitors; D010940:Plant Proteins