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Purification and Characterization of a Cysteine Protease from Sprouted Lablab purpureus Seed Radicle Extract: Its Effect on Blood Coagulation

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Natural Product Experiments in Drug Discovery

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Abstract

Germinating seeds contain several groups of proteases that have been implicated in seed germination. The major function of the proteases is to help in degradation and mobilization of storage proteins. The germinating seeds rich in active enzymes, especially proteases, have been used in therapeutic and as antiaging diet components.

This study aimed to study the role of cysteine protease purified from sprouted Lablab purpureus seed radicle extract (LPRE) in hemostasis and platelet aggregation. The cysteine protease has been purified by two-step purification using Sephadex G-75 and DEAE Sephadex A-50 column chromatography. The protease is named as “Purpurease.” The SDS-PAGE, reversed phase HPLC, and MALDI-TOF mass spectrometry confirmed the homogeneity and purity of the purified protease. The molecular weight of the “Purpurease” was found to be 47.2 kDa. The protease was assayed using fat-free casein and gelatin as substrate. Among protease inhibitors EDTA, 1,10-phenanthroline, aprotinin, E-64, and pepstatin-A, E-64 inactivated the enzyme irreversibly indicating the cysteine type of protease. The protease hydrolyzed fibrinogen in a dose-dependent manner. The protease was evaluated for effect on blood coagulation both in vitro and in vivo studies. The protease showed strong anticoagulant activity in studied methods of prothrombin time (PT), activated partial thromboplastin time (APTT), and recalcification time (RT). In an in vivo study using mouse model, the protease exhibited anticoagulant activity in tail bleeding assay increasing the bleeding time from 140 ± 5 sec to 650 ± 20 sec. The protease further evaluated for its effect on platelet aggregation. The protease inhibited the platelet aggregation significantly when collagen and ADP used as agonists. The protease inhibited platelet aggregation to an extent of 82% and 68% with collagen and ADP-induced aggregation, respectively.

Thus a cysteine protease has been purified and evaluated for its effect on coagulation and platelet aggregation. The protease can be studied further for its therapeutic potential.

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Authors and Affiliations

  1. Department of Studies and Research in Biotechnology, Tumkur University, Tumkur, Karnataka, India

    R. S. Thoyajakshi & D. Poornima

  2. Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, Karnataka, India

    S. Nagaraju

  3. Department of Life Science, JnanaBharathi Campus, Bangalore University, Bangalore, Karnataka, India

    H. Ravi Kumar

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  1. R. S. Thoyajakshi
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  2. S. Nagaraju
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  3. H. Ravi Kumar
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  4. D. Poornima
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Editors and Affiliations

  1. Key Laboratory of Economic Plants and Biotechnology and the Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

    Karuppusamy Arunachalam

  2. Key Laboratory of Economic Plants and Biotechnology and the Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

    Xuefei Yang

  3. Department of Botany, NSS College, Nemmara, Palakkad, Kerala, India

    Sreeja Puthanpura Sasidharan

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Thoyajakshi, R.S., Nagaraju, S., Ravi Kumar, H., Poornima, D. (2023). Purification and Characterization of a Cysteine Protease from Sprouted Lablab purpureus Seed Radicle Extract: Its Effect on Blood Coagulation. In: Arunachalam, K., Yang, X., Puthanpura Sasidharan, S. (eds) Natural Product Experiments in Drug Discovery. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2683-2_19

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  • DOI: https://doi.org/10.1007/978-1-0716-2683-2_19

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