Soybean Trypsin Inhibitor Assay: The Sequence Effect of Adding Reagents, Factors Involved, and Mechanistic Explanations

For assaying trypsin inhibitor activity (TIA) of protein‐based inhibitors, the sequence of adding enzyme, substrate, and inhibitor has profound effects on measured values. This study systematically investigated this reagents’ sequence effect, with regard to factors involved and mechanistic explanations, using raw and toasted soy flours. When the AOCS standard method (Ba 12‐75), which features adding substrates last, was used, TIA values measured varied greatly with not only the pH and Ca 2+ concentration of the premix (the mixture of the first two reagents) and preincubation time, but also extractant, postextraction workup (addition of Tris assay buffer and/or centrifugation), and extract dilution levels. Raw and toasted soy flours showed different TIA changes with these factors. Yet, when the standard method was slightly modified by adding enzyme last, TIA values obtained were unaffected by the premix pH and Ca 2+ concentration, preincubation time, and extract dilution levels, and thus remained constant. Strong interactions of some factors also existed, as exemplified by significant suppression of the reagents’ sequence effect by the Ca 2+ presence in the premix when the premix was neutral and alkaline, but not acidic. These observations are explainable by three proposed mechanisms: limited hydrolysis of the trypsin inhibitor by trypsin in acidic medium according to a reactive site model of the inhibitor, trypsin autolysis in neutral or alkaline medium, and trypsin protection by Ca 2+ . This study provided a strong basis for modifying the standard method. Because the reactive site model of soybean trypsin inhibitors is applicable to many other protein‐based proteinase inhibitors, the reagents’ sequence will affect the results when assaying their activities.