Rapid Release of Protease Inhibitors from Soybeans

Abstract

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and 4 other trypsin inhibitors of low MW isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in seeds and protein extracts of soybean meal, in seedlings, and in the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined 5 low MW protease inhibitors were found to constitute the following percentages of proteins (wt/wt): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2 and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20 and 24 h, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls and roots of 12 day old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 .mu.g; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20 and 24 h, 10, 2.5, 5, 5 and 2.5 .mu.g, respectively; lyophilisate of water in which roots grew for 20 days, 5 .mu.g; 12 day old cotyledons, roots, epicotyls and hypocotyls, 12.5, 100, > 1000 and > 500 .mu.g, respectively. The results indicate that a large amount of protease inhibitors and lectins are released from seeds during the first 8 h of imbibition. Neither lima bean trypsin inhibitor (MW 10,000) nor Kunitz soybean trypsin inhibitor (MW 21,500) showed competitive inhibition in tests with antisera against low MW soybean protease inhibitors.