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Structure and Characterization of Eriphia verrucosa Hemocyanin

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Abstract

Arthropod hemocyanins (Hcs) are a family of large extracellular oxygen-transporting proteins with high molecular mass and hexameric or multi-hexameric molecular assembly. This study reports for the first time the isolation and characterization of the structure of an arthropod hemocyanin from crab Eriphia verrucosa (EvH) living in the Black Sea. Its oligomeric quaternary structure is based on different arrangements of a basic 6 × 75 kDa hexameric unit, and four of them (EvH1, EvH2, EvH3, and EvH4) were identified using ion-exchange chromatography. Subunit 3 (EvH3) shows high similarity scores (75.0, 87.5, 91.7, and 75.0 %, respectively) by comparison of the N-terminal sequence of subunit 1 from Cancer pagurus of the North Sea (Cp1), subunits 3 and 6 of Cancer magister (Cm3 and Cm6), and subunit 2 of Carcinus aestuarii (CaSS2), respectively. Moreover, a partial cDNA sequence (1309 bp) of E. verrucosa hemocyanin encoding a protein of 435 amino acids was isolated. The deduced amino acid sequence shows a high degree of similarity with subunits 3, 4, 5, and 6 of C. magister (81–84 %). Most of the hemocyanins are glycosylated, and three putative O-linkage sites were identified in the partial amino acid sequence of EvH at positions 444–446, 478–480, and 547–549, respectively. The higher stability of native Hc in comparison to its subunit EvH4 as determined by circular dichroism (CD) could be explained with the formation of a stabilizing quaternary structure.

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Acknowledgments

This work was supported by Bulgarian Ministry of Education, projects DHRC /01/6 and “Young researchers” DMU 03/26, Deutsche Forschungsgemeinstchaft (DFG-STE 1819/5-1/2012),Germany, grant no. BG051PO001-3.3.06-0025, financed by the European Social Fund and Operational Programme Human Resources Development (2007–2013) and co-financed by Bulgarian Ministry of Education, Youth and Science.

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Authors and Affiliations

  1. Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, G. Bonchev str. 9, Sofia, 1113, Bulgaria

    A. Dolashki & P. Dolashka

  2. AgroBioInstitute, 8 Dragan Tsankov, Str., 1164, Sofia, Bulgaria

    M. Radkova, E. Todorovska & M. Ivanov

  3. Institute for Cell Biology, Department of Immunology, University of Tübingen, Auf der Morgenstelle 15, D-72076, Tϋbingen, Germany

    S. Stevanovic

  4. CNR-ISTM, Corso Stati Uniti 4, 35129, Padova, Italy

    L. Molin & P. Traldi

  5. Interfacultary Institute of Biochemistry, University of Tϋbingen, Hoppe-Seyler-Str. 4, D-72076, Tϋbingen, Germany

    W. Voelter

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  1. A. Dolashki
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  2. M. Radkova
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  3. E. Todorovska
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  4. M. Ivanov
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  5. S. Stevanovic
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  8. W. Voelter
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  9. P. Dolashka
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Correspondence to P. Dolashka.

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Dolashki, A., Radkova, M., Todorovska, E. et al. Structure and Characterization of Eriphia verrucosa Hemocyanin. Mar Biotechnol 17, 743–752 (2015). https://doi.org/10.1007/s10126-015-9653-9

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  • DOI: https://doi.org/10.1007/s10126-015-9653-9

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