Developing an optimized method for measuring chymotrypsin inhibitor activity in protein products

Abstract

Protease inhibitors of protein nature, such as trypsin inhibitors and chymotrypsin inhibitors, are rich in seeds of legume crops. Soybeans contain Kunitz inhibitor and Bowman-Birk inhibitor. The former mainly inhibits trypsin, while the latter inhibits both trypsin and chymotrypsin. Other legumes contain similar types. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement due to its antinutritional implication. However, Bowman-Birk inhibitor has been shown therapeutic. It is also more resistant to heat than Kunitz inhibitor. As increasing volumes of plant proteins are being used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Yet, reported methods for CIA assay vary greatly, with no standard method being available. Three years ago, at our USDA lab, we developed an improved method for measuring trypsin inhibitor activity, which was later adopted as AOCS Official Method, Ba 12a-2020. This presentation reports our new effort in developing a method for measuring CIA, using N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Unlike the substrate for measuring trypsin inhibitor activity, BTpNA is not water soluble, an organic solvent that is miscible with water must be present. Therefore, the assay system for measuring CIA was much more complicated than that for measuring trypsin inhibitor activity. This made the method development more difficult than originally thought. After investigating effects of many assay parameters, such as organic solvent, the sequence of adding reagents, % chymotrypsin inhibition, etc., an optimized method for CIA measurement was finally developed. It featured dimethylformamide as the organic solvent, the enzyme-last sequence, 5 mL total assay volume, and calculation of the inhibitor activity based on % chymotrypsin inhibition. The proposed method was reliable and robust and could be standardized for measuring CIA in various protein products.