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EXTRACTION, PURIFICATION, AND CHARACTERIZATION OF A TRYPSIN INHIBITOR FROM COWPEA SEEDS (Vigna unguiculata)

Author:
Wang, Jia, Li, Xiaona, Xia, Xunfeng, Li, Hao, Liu, Jing, Li, Qing X., Li, Ji, Xu, Ting
Source:
Preparative biochemistry & biotechnology 2014 v.44 no.1 pp. 1-15
ISSN:
1532-2297
Subject:
Vigna unguiculata, ammonium sulfate, anion exchange chromatography, cowpeas, denaturation, desorption, gel chromatography, genetically modified foods, hexane, ionization, mass spectrometry, molecular weight, pH, polyacrylamide gel electrophoresis, purification methods, risk assessment, seeds, sodium chloride, sodium dodecyl sulfate, trypsin, trypsin inhibitors
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.