title: Extraction, purification, and characterization of a trypsin inhibitor from cowpea seeds (Vigna unguiculata).
abstract: Protease inhibitors against trypsin were extracted from cowpea seeds, purified, and characterized. After the seed powder was defatted with hexane, the cowpea trypsin inhibitor (CpTI) was extracted with 0.15 M NaCl for 30 min. The crude extracts were then heated at 90°C for 10 min, followed by precipitation with 40-65% saturation ammonium sulfate, by which the protein purity increased approximately 15-fold. The CpTI had approximate 88-fold and 186-fold purification after anion-exchange chromatography (Super-Q) and gel filtration (Sephadex G-200), respectively. A broad band of the purified CpTI on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicates a degree of heterogeneity and partial denaturation of CpTI, having a molecular mass of ∼8000 kD. Multiple peaks between 7451 and 8898 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy also suggest heterogeneity. The purified CpTI was stable at 90°C for 60 min, pH 5-10, and 0-3.0% of NaCl. The purification method described here can be used to obtain highly purified CpTI for its studies such as risk assessment of CpTI genetically modified foods.
authors.affiliation: a College of Resources and Environmental Sciences , China Agricultural University , Beijing , China.
authors.forename: Jia
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaona
authors.initials: X
authors.lastname: Li
authors.forename: Xunfeng
authors.initials: X
authors.lastname: Xia
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Qing X
authors.initials: QX
authors.lastname: Li
authors.forename: Ji
authors.initials: J
authors.lastname: Li
authors.forename: Ting
authors.initials: T
authors.lastname: Xu
mesh_terms: D000818:Animals; D002417:Cattle; D007887:Fabaceae / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D010940:Plant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin