Effect of the reactant mixing sequence on the chymotrypsin inhibition assay

In assaying chymotrypsin inhibition by the soybean Bowman-Birk inhibitor, two sequences of mixing the reactants were tried: adding the substrate last (s-last test) or adding the enzyme last (e-last test). The inhibition values obtained from the s-last test were either equal to or lower than those from the e-last test, depending on the pre-mix pH and pre-incubation time, while the values from the e-last test were independent of these conditions. The differences between the two tests were largest at a pre-mix pH of 4.0 or 8.5 and zero near pH 7.0. Abruptly changing the pre-mix pH from 4.0 to 7.0 brought the values of the two tests closer. These observations suggest a reversible, yet limited, hydrolysis of the inhibitor by the very enzyme it inhibits and indicate the greater reliability of the e-last test over the s-last test, paralleling a similar previous finding on the trypsin inhibition assay.