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Phenoloxidase Activity of Helix aspersa Maxima (Garden Snail, Gastropod) Hemocyanin

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Abstract

The oxygen-transporting protein, hemocyanin (Hc), of the garden snail Helix aspersa maxima (HaH) was isolated and kinetically characterized. Kinetic parameters of the reaction of catalytic oxidation of catechol to quinone, catalyzed by native HaH were determined: the V max value amounted to 22 nmol min−1 mg−1, k cat to 1.1 min−1. Data were compared to those reported for other molluscan Hcs and phenoloxidases (POs). The o-diphenoloxidase activity of the native HaH is about five times higher than the activity determined for the Hcs of the terrestrial snail Helix pomatia and of the marine snail Rapana thomasiana (k cat values of 0.22 and 0.25 min−1, respectively). The K m values obtained for molluscan Hcs from different species are comparable to those for true POs, but the low catalytic efficiency of Hcs is probably related to inaccessibility of the active sites to potential substrates. Upon treatment of HaH with subtilisin DY, the enzyme activity against substrate catechol was considerably increased. The relatively high proteolytically induced o-diPO activity of HaH allowed using it for preparation of a biosensor for detection of catechol.

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Abbreviations

DMF:

N,N-dimethylformamide

l-Dopa:

3,4-Dihydroxy-l-phenyl-alanine

EDTA:

Ethylenediaminetetraacetic acid

MBTH:

3-Methyl-2-benzothiazolinone hydrazone

PAGE:

Polyacrylamide gel electrophoresis

PMSF:

Phenylmethanesulfonyl fluoride

SDS:

Sodium dodecyl sulphate

Tris–HCl:

Tris (hydroxymethyl) amino-methane hydrochloride

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Acknowledgments

We are grateful to the National Science Fund of the Ministry of Education, Youth and Science, Bulgaria, for the financial support of a research Grant DTK 02/78.

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Authors and Affiliations

  1. Sofia, Bulgaria

    Yuliana Raynova, Lyuba Doumanova & Krassimira Nikolova Idakieva

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  1. Yuliana Raynova
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  2. Lyuba Doumanova
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  3. Krassimira Nikolova Idakieva
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Correspondence to Krassimira Nikolova Idakieva.

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Raynova, Y., Doumanova, L. & Idakieva, K.N. Phenoloxidase Activity of Helix aspersa Maxima (Garden Snail, Gastropod) Hemocyanin. Protein J 32, 609–618 (2013). https://doi.org/10.1007/s10930-013-9523-0

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  • DOI: https://doi.org/10.1007/s10930-013-9523-0

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