Submitted to: Korean Journal of Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 1, 2004
Publication Date: September 1, 2004
Citation: Krishnan, H.B. 2004. A simple and rapid method to isolate low molecular weight proteinase inhibitors from soybean. Korean Journal of Crop Science. 49:342-348. Interpretive Summary: Soybeans are a rich source of protein. Several studies have demonstrated that diets containing soybean protein contribute to human health beyond the nutritional aspects. Among soybean seed proteins, Bowman-Birk proteinase inhibitor (BBI) has received particular attention because of its potential to prevent cancer. These low molecular weight proteinase inhibitors are traditionally purified by ammonium sulfate precipitation, gel filtration, column chromatography, or high performance liquid chromatography. Each of these procedures is time-consuming and result in limited amounts of purified material. Therefore, establishing a simple protocol for the isolation and purification of these proteins would be beneficial. The results presented in this study show that direct extraction of soybean seed with 60% isopropanol resulted in a fraction that is enriched in the low molecular weight proteinase inhibitors. Since the isopropanol extraction procedure is very simple and results in large amounts of the BBI-enriched fraction, it could be the extraction method of choice for scientists contemplating epidemiological and laboratory studies dealing with carcinogenesis.
Technical Abstract: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 60% isopropanol extract of soybean (Glycine max [L.] Merr.) seed revealed two abundant proteins with molecular masses of 19 and 10 kDa. Amino acid analysis revealed that the isopropanol-extractable fraction was rich in cysteine. Two-dimensional gel electrophoretic analysis indicated that the 19 kDa and 10 kDa proteins had pI of 4.2 and 4.0 respectively. Peptide mass fingerprints of trypsin digests of the two proteins obtained using matrix-assisted, laser desorption/ionization-time of flight mass spectroscopy revealed the 19 kDa protein was Kunitz trypsin inhibitor and the 10 kDa protein was Bowman-Birk proteinase inhibitor. Under non-denaturing conditions, the isopropanol-extracted proteins inhibited trypsin and chymotrypsin activity. Results presented in this study demonstrate that isopropanol extraction of soybean seed could be used as a simple and rapid method to obtain a protein fraction enriched in Kunitz trypsin and Bowman-Birk proteinase inhibitors. Since proteinase inhibitors are rich in sulfur amino acids and are putative anticarcinogens, their rapid and inexpensive isolation by isopropanol could facilitate efforts in nutrition and cancer research.