, Volume 230, Issue 2, pp 429–439 | Cite as

Multiplicity of aspartic proteinases from Cynara cardunculus L.

  • Ana Cristina Sarmento
  • Henrique Lopes
  • Cláudia S. Oliveira
  • Rui Vitorino
  • Bart Samyn
  • Kjell Sergeant
  • Griet Debyser
  • Jozef Van Beeumen
  • Pedro Domingues
  • Francisco Amado
  • Euclides Pires
  • M. Rosário M. Domingues
  • Marlene T. Barros
Original Article


Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO2)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.


Aspartic proteinases Mass spectrometry Protein characterisation Specificity 



Aspartic proteinase


Liquid chromatography


Matrix-assisted laser desorption/ionisation


Mass spectrometry


Time of flight


Trifluoroacetic acid


Ultra-pure water





This research work was financed by POCI/QUI/60791/2004. AC Sarmento, R Vitorino and CS Oliveira were supported by Fundação para a Ciência e Tecnologia (grant nos. BPD/38008/2007, BPD/14968/2004, BPD/26670/2006). B. Samyn is a postdoctoral fellow of the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen, Belgium).

Supplementary material

425_2009_948_MOESM1_ESM.doc (180 kb)
Supplementary material 1 (DOC 180 kb)


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Ana Cristina Sarmento
    • 1
  • Henrique Lopes
    • 2
  • Cláudia S. Oliveira
    • 1
  • Rui Vitorino
    • 2
  • Bart Samyn
    • 3
  • Kjell Sergeant
    • 3
    • 4
  • Griet Debyser
    • 3
  • Jozef Van Beeumen
    • 3
  • Pedro Domingues
    • 2
  • Francisco Amado
    • 2
  • Euclides Pires
    • 5
  • M. Rosário M. Domingues
    • 2
  • Marlene T. Barros
    • 5
    • 6
  1. 1.CESAM, Department of BiologyUniversity of AveiroAveiroPortugal
  2. 2.Department of ChemistryUniversity of AveiroAveiroPortugal
  3. 3.Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein EngineeringGhent UniversityGhentBelgium
  4. 4.Department of Environment and AgrobiotechnologiesCRP-GLBelvauxLuxembourg
  5. 5.Center for Neuroscience and Cell BiologyIBILICoimbraPortugal
  6. 6.Instituto de Ciências da SaúdeUniversidade Católica PortuguesaViseuPortugal

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