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.
LIU
K S
KS
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824-1224.
Markakis
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P
D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives
D007202:Indicators and Reagents; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014443:Tyrosine; C022787:benzoyltyrosine 4-nitroanilide; D002918:Chymotrypsin