Method for Determining Chymotrypsin Inhibitor Activity: Investigations Into Sample Blank Measurements, Factors Involved, and Further Improvement

ABSTRACT An optimized method for determining chymotrypsin inhibitor activity (CIA) for legume and cereal products was recently developed. As a follow‐up, the present study focused on sample blank (SB) measurement and further method improvement for the previously developed CIA assay. It started with an observation that the reagent addition sequence (RAS) had profound effects on SB with soybean samples but not with purified inhibitors. Several hypotheses were proposed, including that the effect of RAS on SB results from differential interactions between other components (including other proteins) in sample extracts with those in the substrate working solution in the presence and absence of acetic acid. Several factors were investigated for their effects and interactions with RAS on SB measurements. These included the duration between adding the second and third reagents, the absorbance at each step of the measurement, the effects of presence and absence of calcium (Ca 2+ ), Ca 2+ concentration, method of sample extraction and post‐extraction storage, post‐reaction centrifugation, wavelength for absorbance measurement, sample type, and volumes of diluted sample extracts. The experiments validated the hypotheses and showed that among the different RAS, the sequence of adding Inhibitor, Acid, Substrate and Enzyme (the IASE sequence) was least affected by most factors and overnight refrigeration of sample extracts had little effect. It was concluded that the RAS effect on SB was unavoidable. Recommendations include using an assay buffer containing 20 mM Ca 2+ , using the IASE sequence with 0.8 mL sample volume for SB measurements, and centrifuging all reaction mixtures before absorbance measurements.