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

Authors: Liu, Keshun

Submitted to: Sustainable Food Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2023
Publication Date: 3/27/2023
Citation: Liu, K. 2023. Chymotrypsin inhibitor assay: Expressing, calculating, and standardizing inhibitory activity in absolute amounts of chymotrypsin inhibited. Sustainable Food Proteins. 1(1):30-44. https://doi.org/10.1002/sfp2.1004.
DOI: https://doi.org/10.1002/sfp2.1004

Interpretive Summary: Protease inhibitors of proteinaceous nature are ubiquitously distributed in the plant, animal, and microbial kingdoms. They are particularly rich in legume seeds. In soybeans, two types of protease inhibitors have been identified: Kuniz trypsin inhibitor and Bowman-Birk trypsin and chymotrypsin inhibitor. Both are all considered antinutritional. Historically, trypsin inhibitor activity has been primarily measured. However, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in soybeans and other legume products. Not long ago, USDA researchers at Aberdeen, Idaho, developed an optimized method that can accurately measure CIA. However, like most previously reported methods, the new method expresses CIA as chymotrypsin units inhibited/mg sample. Although this arbitrary unit of expression is easy to define, it gives values that vary with methods. Furthermore, beside the arbitrary unit, CIA has also been expressed in other units. This variation in CIA expression units has made it more difficult to compare results among studies. For standardizing and unifying units for expressing CIA, the same USDA researchers at Aberdeen, Idaho, carried out a continuous study, with an objective to express CIA in absolute amounts of chymotrypsin inhibited, by determining a conversion factor between arbitrary unit and µg chymotrypsin inhibited and standardizing it with a reference chymotrypsin. The new method can now express results in mg chymotrypsin inhibited/g sample by dividing the arbitrary unit by 1.5, which is the standardized conversion factor determined between the two units. Furthermore, if another method or any future methods for measuring CIA follow the same concept of standardization against the same reference chymotrypsin, it could be possible to compare CIA results among studies.

Technical Abstract: There 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 'A400) = 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.