Soybean trypsin inhibitor assay: the sequence effect of adding three reagents and factors involved

Authors: Liu, Keshun

Submitted to: American Oil Chemists' Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 10/25/2017
Publication Date: 11/10/2017
Citation: Liu, K. 2017. Soybean trypsin inhibitor assay: the sequence effect of adding three reagents and factors involved. American Oil Chemists' Society Meeting, Nov. 10-12, 2017, Wuxi, China. S6No2.

Interpretive Summary:

Technical Abstract: Much like proteases, protease inhibitors of a protein nature are ubiquitously distributed in the plant, animal and microbial kingdoms. They play key regulatory roles in many biological processes. Trypsin inhibitors (TI) are a key anti-nutritional factor in soybeans and other legume products, since their inactivation by heat treatments parallels the improvement in nutritive values and growth performance when fed to animals. Therefore, TI activity has been an important quality parameter. There are many reported methods for assaying TI activity, but most are colorimetric, resulting from a cleaving action of trypsin on a synthetic chromogenic substrate, benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPA), and based on a difference in absorbance at 410 nm between the absence and the presence of an inhibitor. In 1970’s, the American Association of Cereal Chemists and the American Oil Chemists’ Society officially approved a same standard method. It features mixing an inhibitor sample in a range of concentrations with trypsin followed by adding the substrate BAPA to start the color reaction (hereby termed the S-last test), since the pre-incubation of inhibitor with enzyme is commonly believed necessary to permit full formation of an enzyme-inhibitor complex and obtain equilibrium data. Since then, some researchers have showed that, in contrast to the common belief, mixing the inhibitor with the substrate followed by enzyme addition to start the reaction (hereby termed the E-last test) tends to give higher inhibition value than the S-last test at certain pre-incubation pH ranges. The sequence effect was attributed to the limited hydrolysis of the inhibitor by the very enzyme it inhibits. Others suggested use of a single inhibitor concentration over a range of levels. However, the official method has remained unchanged, with repeated re-approval by the two societies over the years. To address this important issue, at the U.S. Department of Agriculture we recently conducted a study on analyzing TI activity in soybeans using the 10 mL assay system specified by the standard method. The objectives were to compare results obtained with the S-last test with those from the E-last test and to investigate factors affecting the sequence effect. New results not only confirmed the sequence effect, but also showed that, in addition to pre-incubation pH and time, extracting solvents, post extraction treatments, and the sample dilution factors all had significant effects. More specifically, with a same level of soybean TI, the E-last test generated constant inhibition results independent of these factors while the S-last test gave results varying with these factors. Thus, in assaying TI activity of a protein nature, the E-last test should be used.