Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

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

References

  • Albalasmeh AA, Berhe AA, Ghezzehei TA (2013) A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry. Carbohydr Polym 97(2):253–261. doi:10.1016/j.carbpol.2013.04.072

    Article  CAS  Google Scholar 

  • Antao CM, Malcata FX (2005) Plant serine proteases: biochemical, physiological and molecular features. Plant Physiol Biochem 43(7):637–650. doi:10.1016/j.plaphy.2005.05.001

    Article  CAS  Google Scholar 

  • Asif-Ullah M, Kim KS, Yu YG (2006) Purification and characterization of a serine protease from Cucumis trigonus Roxburghi. Phytochemistry 67(9):870–875. doi:10.1016/j.phytochem.2006.02.020

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Casano LM, Desimone M, Trippi VS (1989) Proteolytic activity at alkaline pH in oat leaves isolation of an aminopeptidase. Plant Physiol 91(4):1414–1418

    Article  CAS  Google Scholar 

  • Condrea E, Yang CC, Rosenberg P (1983) Anticoagulant activity and plasma phosphatidylserine hydrolysis by snake venom phospholipases A2. Thromb Haemost 49(2):151

    CAS  Google Scholar 

  • Deepak S, Shailasree S, Kini RK, Hause B, Shetty SH, Mithöfer A (2007) Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola. Planta 226(2):323–333. doi:10.1007/s00425-007-0484-4

    Article  CAS  Google Scholar 

  • Devaraja S, Nagaraju S, Mahadeswaraswamy YH, Girish KS, Kemparaju K (2008) A low molecular weight serine protease: purification and characterization from Hippasa agelenoides (funnel web) spider venom gland extract. Toxicon 52(1):130–138. doi:10.1016/j.toxicon.2008.04.168

    Article  CAS  Google Scholar 

  • Kaneda M, Tominaga N (1975) Isolation and characterization of a proteinase from the sarcocarp of melon fruit. J Biochem 78(6):1287–1296

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  Google Scholar 

  • TK Lim (2012) Edible medicinal and non-medicinal plants. Fruits, Vol 2. Springer, Dordrecht

  • Manjula B, Swamy YHM, Devaraja S, Girish KS, Kemparaju K (2015) Clot promoting and dissolving properties of cucumber (Cucumis sativus) sap, validating its use in traditional medicine. 2015:8

  • Nafeesa Z, Shivalingu BR, Vivek HK, Priya BS, Swamy SN (2015) Exploring a new serine protease from Cucumis sativus L. Appl Biochem Biotechnol 175(6):2787–2794. doi:10.1007/s12010-014-1462-5

    Article  CAS  Google Scholar 

  • Nema NK, Maity N, Sarkar B, Mukherjee PK (2011) Cucumis sativus fruit-potential antioxidant, anti-hyaluronidase, and anti-elastase agent. Arch Dermatol Res 303(4):247–252

    Article  CAS  Google Scholar 

  • Pande M, Dubey VK, Yadav SC, Jagannadham MV (2006) A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization. J Agric Food Chem 54(26):10141–10150. doi:10.1021/jf062206a

    Article  CAS  Google Scholar 

  • Rahman AHMM (2013) Ethano-medico-botanical investigation on cucurbits of the Rajshahi Division. Bangladesh J Medicinal Plants Studies 1(3):118–125

    Google Scholar 

  • Rajesh R, Raghavendra Gowda CD, Nataraju A, Dhananjaya BL, Kemparaju K, Vishwanath BS (2005) Procoagulant activity of Calotropis gigantea latex associated with fibrin(ogen)olytic activity. Toxicon 46(1):84–92. doi:10.1016/j.toxicon.2005.03.012

    Article  CAS  Google Scholar 

  • Rajesh R, Nataraju A, Gowda CD, Frey BM, Frey FJ, Vishwanath BS (2006) Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot. Biochimie 88(10):1313–1322. doi:10.1016/j.biochi.2006.06.007

    Article  CAS  Google Scholar 

  • Shivalingu BR, Vivek HK, Nafeesa Z, Priya BS, Swamy SN (2015) Comparative analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of turmeric species. J Ethnopharmacol 172:261–264. doi:10.1016/j.jep.2015.06.018

    Article  CAS  Google Scholar 

  • Sotiroudis G, Melliou E, Sotiroudis TG, Chinou I (2010) chemical analysis, antioxidant and antimicrobial activity of three Greek cucumber (Cucumis sativus) cultivars. J Food Biochem 3 (s1):61–78

    Article  Google Scholar 

  • Torres-Urrutia C, Guzman L, Schmeda-Hirschmann G, Moore-Carrasco R, Alarcon M, Astudillo L, Gutierrez M, Carrasco G, Yuri JA, Aranda E, Palomo I (2011) Antiplatelet, anticoagulant, and fibrinolytic activity in vitro of extracts from selected fruits and vegetables. Blood Coagul Fibrinolysis 22(3):197–205. doi:10.1097/MBC.0b013e328343f7da

    Article  Google Scholar 

  • Uchikoba T, Horita H, Kaneda M (1990) Proteases from the sarcocarp of yellow snake-gourd. Phytochemistry 29(6):1879–1881

    Article  CAS  Google Scholar 

  • Uchikoba T, Yonezawa H, Kaneda M (1998) Cucumisin like protease from the sarcocarp of Benincasa hispida var. Ryukyu. Phytochemistry 49(8):2215–2219

    Article  CAS  Google Scholar 

  • Venkatesh BK, Achar RR, Sharanappa P, Priya BS, Swamy SN (2015) Synergistic caseinolytic activity and differential fibrinogenolytic action of multiple proteases of Maclura spinosa (Roxb. ex Willd.) latex. Pharmacogn Mag 11(Suppl 3):S457–S461. doi:10.4103/0973-1296.168987

    CAS  Google Scholar 

  • Vishwanath BS, Kini RM, Gowda TV (1987) Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid. Toxicon 25(5):501–515

    Article  CAS  Google Scholar 

  • Wilimowska-Pelc A, Polanowski A, Kolaczkowska MK, Wieczorek M, Wilusz T (1982) Aspartyl proteinase from cucumber (Cucumis sativus) seeds. Prepration and characterstics. Acta Biochimica Polonica 30(1):23–31

    Google Scholar 

  • Yamaguchi T, Yamashita Y, Takeda I, Kiso H (1982) Proteolytic enzymes in green asparagus, kiwi fruit and miut: occurrence and partial characterization. Agric Biol Chem 46(8):1983–1986. doi:10.1080/00021369.1982.10865376

    CAS  Google Scholar 

  • Yariswamy M, Shivaprasad HV, Joshi V, Nanjaraj Urs AN, Nataraju A, Vishwanath BS (2013) Topical application of serine proteases from Wrightia tinctoria R. Br. (Apocyanaceae) latex augments healing of experimentally induced excision wound in mice. J Ethnopharmacol 149(1):377–383. doi:10.1016/j.jep.2013.06.056

    Article  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Science and Technology University, JSS Technical Institutions Campus, Mysuru, Karnataka, 570 006, India

    Zohara Nafeesa, B. R. Shivalingu, K. N. Neema, B. K. Venkatesh & S. Nanjunda Swamy

  2. JSS Research Foundation, JSS Technical Institutions Campus, Mysore, Karnataka, 570 006, India

    Zohara Nafeesa, B. R. Shivalingu, K. N. Neema, B. K. Venkatesh & S. Nanjunda Swamy

  3. Biochemistry Division, Faculty of Life Sciences, JSS University, Mysore, Karnataka, 570 015, India

    Raghu Ram Achar

  4. Liveon Biolabs Pvt. Ltd., Tumakuru, Karnataka, 572106, India

    Veeresh Hanchinal

  5. Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, Karnataka, 570 006, India

    B. S. Priya

Authors
  1. Zohara Nafeesa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B. R. Shivalingu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. N. Neema
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raghu Ram Achar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B. K. Venkatesh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Veeresh Hanchinal
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. B. S. Priya
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S. Nanjunda Swamy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. Nanjunda Swamy.

Ethics declarations

Conflict of interest

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nafeesa, Z., Shivalingu, B.R., Neema, K.N. et al. Procoagulant serine glycoprotease from Cucumis sativus L.: action on human fibrinogen and fibrin clot. 3 Biotech 7, 96 (2017). https://doi.org/10.1007/s13205-017-0686-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-017-0686-9

Keywords

Search

Navigation