Trypsin inhibitor assay: expressing, calculating, and standardizing inhibitor activity in absolute amounts of trypsin inhibited or trypsin inhibitors

Authors: Liu, Keshun

Submitted to: Journal of the American Oil Chemists' Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2021
Publication Date: 4/6/2021
Citation: Liu, K. 2021. Trypsin inhibitor assay: expressing, calculating and standardizing inhibitor activity in absolute amounts of trypsin or trypsin inhibitors. Journal of the American Oil Chemists' Society. 98:355-373. https://doi.org/10.1002/aocs.12475.
DOI: https://doi.org/10.1002/aocs.12475

Interpretive Summary: Trypsin inhibitors (TI) are ubiquitously distributed in the plant, animal and microbial kingdoms. In the legume seeds, their presence is relatively abundant. For many years, TI were considered antinutritional, but lately some of them have been found beneficial to humans and animals by exerting bioactive, therapeutic, or immune-therapeutic effects. Among different methods for measuring TI activity in various samples, three units have been used for expressing measured results, including an arbitrary unit as trypsin unit inhibited, absolute amounts of trypsin inhibited, and absolute amounts of trypsin inhibitors. This makes comparison of results among studies difficult and in some cases impossible. With increasing use of plant proteins for food or feed, it becomes increasingly important to have a common unit that is accurate in expressing results, easy to understand, and comparable among studies using different assay methods. For addressing the problem with expression units, USDA-ARS researchers at Aberdeen, Idaho, recently conducted a study, using an improved TI assay method developed earlier in the same ARS lab. The study involved developing new approaches to determine conversion factors between different units and standardizing the conversion factors against a reference trypsin based on specific activity of the enzyme. When measured TI activity is expressed in absolute amounts of trypsin inhibited and standardization is applied to other methods, comparison of among methods will become possible.

Technical Abstract: For expressing trypsin inhibitor activity (TIA), trypsin units inhibited (TUI), trypsin inhibited, and trypsin inhibitors have been used. Although the last two units are preferred, their calculations in current practices require refinement. With the proposed AOCS method Ba 12a-2020, four experiments were conducted, using four trypsin preparations having specific activity of 11,625, 12,602, 13,728, and 14,926 Na-benzoyl-L-arginine ethyl ester (BAEE) units/mg protein, respectively. Experiment 1 determined the relationship between absorbance at 410'nm (A410) and trypsin concentration. Experiment 2 involved assaying raw and heated soybeans, expressing TIA as TUI/mg sample and µg trypsin inhibited/mg sample, and determining conversion factors between the two units. Experiment 3 resembled Experiment 2 except for using purified soybean Kunitz inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Conversion factors determined correlated highly with trypsin-specific activity (R2 = 0.9789). After standardizing against a reference trypsin having 15,000 BAEE units/mg protein, a standardized conversion factor of 0.03 A410 (1.5 TUI) = 1 µg trypsin inhibited was determined. It remained consistent regardless of trypsin specific activity, with or without inhibitors, and type of inhibitor samples. By using purified inhibitors (Experiment 3), conversion values between TUI and µg trypsin inhibitor and between µg trypsin inhibited and µg trypsin inhibitor could also be calculated, enabling expression of TIA in amounts of pure KTI, BBI or their equivalents. Furthermore, when the AOCS method was modified with half substrate concentration, half trypsin concentration or half both (Experiment 4), TIA values in TUI could change with modifications but values in mg trypsin inhibited (standardized) or trypsin inhibitor remained consistent.