Bowman-Birk inhibitors in soybean seed coats

Soybean Bowman-Birk inhibitor (BBI), possessing a molecular weight (MW) of 8 kD, is a known cancer chemopreventative and anticarcinogenic agent Aqueou s extracts of soybean seed coats, rigorously screened to remove cotyledons, contained peroxidase along with three unknown proteins each of which posse ssed an estimated MW of < 10 kD when compared with known standards subjecte d to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because these extracts tested positively for trypsin inhibitor (TI) activit y, our objective was to isolate and characterize the low MW inhibitor(s). T o eliminate the solubilization of peroxidase from soy seed coats, we applie d the classic methodology for BBI isolation which utilizes 60% ethanol. Soy bean 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 extra cts of the alcohol extracted residues provided an additional 4.55% solids w ith 2.71% nitrogen and 27 mg TI/g solids. The protein components in the 60% ethanol solubles were further concentrated by acetone precipitation. The a cetone precipitates and water extractables were each fractionated on a CM S ephadex C-25 column eluted with a salt gradient. The consequent column frac tions from the acetone precipitates when subjected to SDS-PAGE yielded thre e proteins, one of which possessed the mobility similar to a commercially a vailable soybean BBI. Because BBI can simultaneously inhibit both trypsin a nd 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 stai n technique applied to native PAGE of the TI and CTI-active fraction from a cetone precipitates showed evidence for seven isoforms of BBI. Two CM Sepha dex 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 stand ards 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 C TI activity relative to CTI in a commercial BBI standard was 61.9%, whereas , the CTI activity in fractions of column chromatographed water extracts th at 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. (C) 2001 Elsevier Science B.V. All rights reserved.