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3 Biotech

, 7:96 | Cite as

Procoagulant serine glycoprotease from Cucumis sativus L.: action on human fibrinogen and fibrin clot

  • Zohara Nafeesa
  • B. R. Shivalingu
  • K. N. Neema
  • Raghu Ram Achar
  • B. K. Venkatesh
  • Veeresh Hanchinal
  • B. S. Priya
  • S. Nanjunda SwamyEmail author
Original Article
  • 64 Downloads

Abstract

Upon examination of the fruit extract of Cucumis sativus L. for its pharmacological benefits, it was previously observed that it has potential proteolytic, fibrinogenolytic and procoagulant activities. These properties can be attributed to the presence of the protease. In this regard, the present study comprised of purification and characterization of protease. Purification of the enzyme involved ammonium sulfate precipitation followed by gel filtration and ion exchange chromatography. The purified cucumis protease (CPro) exhibits homogeneity as attested by SDS-PAGE and RP-HPLC with a retention time of 14.246 min with molecular mass ~75.3 kDa. CPro was identified as a glycoprotein and serine protease. Azocasein is the preferred substrate for CPro as it showed low Km value of 0.3809 mg/ml. Purified CPro exhibits optimum activity at 37 °C and pH 8. CPro shows its involvement in hemostasis—the very first step in wound healing. CPro degrades the subunits of human fibrinogen in the order Aα > Bβ > γ. It also hydrolyzes the subunits of the partially cross-linked fibrin clot in the order α-polymer > γ–γ dimer > β-chain. CPro reduced the clotting time of citrated plasma, prothrombin time and activated partial thromboplastin time of plasma. CPro is neither hemorrhagic nor edema-inducing, thus considered to be a non-toxic protease. This work provides evidence for the use of cucumber extract in wound healing and authenticates its use in cosmetics.

Keywords

Serine protease Glycoprotein Fibrin(ogen)olytic Hemostasis Procoagulant Cucumis protease 

Abbreviations

CPro

Cucumis protease

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

RP-HPLC

Reverse-phase high-performance liquid chromatography

AqFEC

Aqueous fruit extract of European cucumber

RFC

Relative clotting factor

PMSF

Phenyl methyl sulphonyl fluoride

EDTA

Ethylene diamine tetra acetic acid

EGTA

Ethylene glycol-bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid

IAA

Iodoacetic acid

PT

Prothrombin time

APPT

Activated partial thromboplastin time

TFA

Trifluoroacetic acid

PAS

Periodic acid-Schiff

Notes

Acknowledgements

ZN would like to express gratitude to the University Grant Commission, New Delhi for financial assistance under Maulana Azad National Fellowship (F1-17.1/2013-14/MANF-2013-14-MUS-KAR-20358).

Compliance with ethical standards

Conflict of interest

The authors confirm that this article content has no conflict of interest.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Zohara Nafeesa
    • 1
    • 2
  • B. R. Shivalingu
    • 1
    • 2
  • K. N. Neema
    • 1
    • 2
  • Raghu Ram Achar
    • 3
  • B. K. Venkatesh
    • 1
    • 2
  • Veeresh Hanchinal
    • 4
  • B. S. Priya
    • 5
  • S. Nanjunda Swamy
    • 1
    • 2
    Email author
  1. 1.Department of Biotechnology, Sri Jayachamarajendra College of EngineeringJSS Science and Technology UniversityMysuruIndia
  2. 2.JSS Research FoundationMysoreIndia
  3. 3.Biochemistry Division, Faculty of Life SciencesJSS UniversityMysoreIndia
  4. 4.Liveon Biolabs Pvt. Ltd.TumakuruIndia
  5. 5.Department of Studies in ChemistryUniversity of MysoreMysoreIndia

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