Bowman–Birk inhibitors in soybean seed coats

Abstract

Soybean Bowman–Birk inhibitor (BBI), possessing a molecular weight (MW) of 8 kD, is a known cancer chemopreventative and anticarcinogenic agent Aqueous extracts of soybean seed coats, rigorously screened to remove cotyledons, contained peroxidase along with three unknown proteins each of which possessed an estimated MW of <10 kD when compared with known standards subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because these extracts tested positively for trypsin inhibitor (TI) activity, our objective was to isolate and characterize the low MW inhibitor(s). To eliminate the solubilization of peroxidase from soy seed coats, we applied the classic methodology for BBI isolation which utilizes 60% ethanol. Soybean seed coats, which represent 8% of the whole seed, were defatted, then twice extracted with 60% aqueous ethanol to yield 6.96% sugary solids with 1.56% Kjeldahl nitrogen and a TI activity of 44 mg TI/g solids. Water extracts of the alcohol extracted residues provided an additional 4.55% solids with 2.71% nitrogen and 27 mg TI/g solids. The protein components in the 60% ethanol solubles were further concentrated by acetone precipitation. The acetone precipitates and water extractables were each fractionated on a CM Sephadex C-25 column eluted with a salt gradient. The consequent column fractions from the acetone precipitates when subjected to SDS-PAGE yielded three proteins, one of which possessed the mobility similar to a commercially available soybean BBI. Because BBI can simultaneously inhibit both trypsin and chymotrypsin, we verified the presence of BBI in those column fractions that eluted after 0.21M NaCl concentration based on colorimetric assays for TI and chymotrypsin inhibitor (CTI) activities. Chymotrypsin negative stain technique applied to native PAGE of the TI and CTI-active fraction from acetone precipitates showed evidence for seven isoforms of BBI. Two CM Sephadex column fractions eluting prior to 0.21 M salt concentration showed two low MW proteins (<7 kD) when stained for protein and compared with MW standards subjected to SDS-PAGE. Based on the summation of CTI active components in the CM Sephadex C-25 column fractions from 60% ethanol extract, their CTI activity relative to CTI in a commercial BBI standard was 61.9%, whereas, the CTI activity in fractions of column chromatographed water extracts that eluted after 0.21M salt concentration was 86.1%. Soybean seed coats may provide a new, inexpensive source of BBI concentrate for use as a potential anticancer agent.