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Purification, characterization and fibrino(geno)lytic activity of cysteine protease from Tabernaemontana divaricata latex

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Abstract

Protease was isolated and purified from Tabernaemontana divaricata latex and its hemostatic potential was analyzed. Crude latex enzyme was purified through ion exchange and gel filtration chromatography. Purified protease was characterized and its thrombin-like (coagulant assay, fibrinogen polymerizing, and fibrinogenolytic activity) and plasmin-like (blood and plasma clot lysis) activities were assessed accordingly. The homogeneous nature of protease was confirmed with the identification of a single band approximately at 25-kDa molecular weight position. The purified enzyme showed an enhancement of 77.32% in clot inducing ability and 89.86% improvement in blood clot lysis in comparison to that by the crude enzyme. All three subunits (Aα, Bβ and γ chains) of human fibrinogen were hydrolyzed by the purified enzyme. PAGE results of the fibrinolytic activity and blood clot lytic effect by the purified enzyme indicated the plasmin-like activity. The study lays a foundation for the development of enzyme-based approaches for pharmaceutical innovations, in which plant latex proteases can be utilized as a potential natural agent for wound healing applications.

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Acknowledgements

The authors thank Prof. Leela Iyengar, Adjunct Professor, Jain (Deemed to be University) for her valuable suggestions and fruitful discussions during the study. We are equally thankful to Dr. Krishna Venkatesh (Former Director at Centre for Emerging Technologies, Jain University), for his help and support. Financial assistance to Dr. Maheshwari Kumari Singh in the form of DST INSPIRE Fellowship, Government of India during the course of study is gratefully acknowledged. Dr. Maheshwari Kumari Singh is also thankful to Hon’ble Chairman and Member Secretary, Karnataka State Pollution Control Board, Govt. of Karnataka, Bangalore for their support.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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  1. Maheshwari Kumari Singh

    Present address: Karnataka State Pollution Control Board, Bangalore, Karnataka, India

Authors and Affiliations

  1. Department of Biochemistry, School of Sciences-Block 1, Jain (Deemed to be University), 9th Main, 18th Cross, Jayanagar 3rd Block, Bangalore, Karnataka, India

    Maheshwari Kumari Singh, Anusha Rajagopalan, Habibu Tanimu & Bindhu Omana Sukumaran

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  1. Maheshwari Kumari Singh
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  2. Anusha Rajagopalan
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  3. Habibu Tanimu
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  4. Bindhu Omana Sukumaran
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Contributions

MKS: data curation; formal analysis; investigation; methodology; project administration; resources; validation; visualization; roles/writing—original draft. AR: roles/writing—original draft; writing—review and editing. HT: writing—review and editing. BOS: conceptualization; formal analysis; investigation; methodology; project administration; resources; software; supervision; validation; visualization; roles/writing—original draft; writing—review and editing.

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Correspondence to Bindhu Omana Sukumaran.

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Conflict of interest

One of the authors Maheshwari Kumari Singh has received Senior Research Fellowship under DST INSPIRE Fellowship during the course of her Ph.D. All the other authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants (who donated blood for recalcification, whole blood assay and blood clot lysis assay-based experiment) included in the study. Fresh human blood samples were collected from healthy volunteers after obtaining the consent in a format approved by Institutional Ethics Committee of Sapthagiri Hospital, Hesaraghatta, Bangalore vide No.SH/ECC/Ph.D./14987 dt: 12.03.2012.

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Singh, M.K., Rajagopalan, A., Tanimu, H. et al. Purification, characterization and fibrino(geno)lytic activity of cysteine protease from Tabernaemontana divaricata latex. 3 Biotech 11, 106 (2021). https://doi.org/10.1007/s13205-021-02643-9

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