Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from (Benth) Seeds.

Introduction: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest.

Objectives: This work aimed to purify a trypsin inhibitor from seeds (TI), describing its kinetic mechanism and anticoagulant effect.

Methods: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. assays were performed to verify its ability to prolong blood clotting time.

Results: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. TI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and TI showed similarity to several Kunitz-type trypsin inhibitors. TI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10 M) and Ki (1.05 x 10 M). Additionally, TI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. TI showed an anticoagulant effect at a concentration of 33 μM, prolonging clotting time by 2.6 times.

Conclusion: Our results suggest that TI can be a biological tool to be used in blood clotting studies.