Chymotrypsin inhibitor assay: Expressing, calculating, and standardizing inhibitory activity in absolute amounts of chymotrypsin inhibited

Abstract

AbstractThere is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in soybean and other legume products, although trypsin inhibitor activity has been primarily measured. Recently, for accurately measuring CIA, an optimized method was developed in our laboratory and published elsewhere. Like most reported methods, the new method expresses CIA as chymotrypsin units (CU) inhibited (CUI)/mg sample. This arbitrary unit makes comparison of results impossible among different methods. The present study solved this problem by expressing CIA in absolute amounts of chymotrypsin inhibited (CId) and standardizing against a reference chymotrypsin. With the new method, two experiments were conducted, using four chymotrypsin reagents having different specific activities (SA), respectively. Experiment 1 determined the relationship between absorbance at 400 nm (A400) and μg chymotrypsin. Experiment 2 measured raw and heated soybeans, expressed CIA as CUI/mg sample and μg CId/mg sample, respectively, and determined conversion factors between the two units. Conversion factors determined in both experiments matched each other but varied with chymotrypsin SA. Yet, after standardizing against the reference chymotrypsin having a fixed SA of 150 N‐benzoyl‐L‐tyrosine‐p‐nitroanilide (BTNA) (equivalent to 50 N‐benzoyl‐L‐tyrosine ethyl ester [BTEE]) units/mg protein, a standardized conversion factor of 1.5 CUI (= 0.015 ΔA400) = 1 μg CId was determined. It remained consistent regardless of chymotrypsin SA, sample heating history, and levels. The new method can now express results in μg CId/mg sample (or mg/g) by dividing CUI/mg sample by 1.5. When other methods follow the same strategies for determining standardized conversion factors, CIA results could be comparable.