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


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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.

Objective:This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), 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. In vitro 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. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100 ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times.

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

Sobre autores

Renato Roma

Department of Biochemistry and Molecular Biology, Federal University of Ceará

Email: info@benthamscience.net

Lucas Dias

Paulista School of Medicine, Support Foundation for the Federal University of São Paulo

Email: info@benthamscience.net

Ana Santos

Agrarian and Biodiversity Sciences Center, Federal University of Cariri

Email: info@benthamscience.net

Romério Silva

Department of Biochemistry and Molecular Biology, Federal University of Ceará

Email: info@benthamscience.net

Maria Santos

Department of Biochemistry and Molecular Biology, Federal University of Ceará

Email: info@benthamscience.net

Bruno Rocha

Department of Biochemistry and Molecular Biology, Federal University of Ceará

Email: info@benthamscience.net

Rômulo Carneiro

Department of Fisheries Engineering, Universidade Federal do Ceará

Email: info@benthamscience.net

Celso Nagano

Department of Fisheries Engineering, Universidade Federal do Ceará

Email: info@benthamscience.net

Alexandre Sampaio

Department of Fisheries Engineering, Universidade Federal do Ceará

Email: info@benthamscience.net

Maria Oliva

Department of Biochemistry, Federal University of São Paulo

Email: info@benthamscience.net

Cláudio Silva

Medical School, Federal University of Cariri

Email: info@benthamscience.net

Racquel Souza

Medical School, Federal University of Cariri

Email: info@benthamscience.net

Claudener Teixeira

Agrarian and Biodiversity Sciences Center, Federal University of Cariri

Autor responsável pela correspondência
Email: info@benthamscience.net

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