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Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species

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Abstract

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase, which play crucial roles in the innate immune response of mollusks. In this research, POs were studied in cultured mollusk species, including scallop Chlamys farreri, abalone Haliotis discus hannai and clam Scapharca subcrenata. The POs were isolated from hemocytes using lineargradient native-PAGE combined with catechol staining. The PO activities and their characters were investigated. The molecular mass of PO in C. farreri was 576 kDa, and it was 228 kDa in H. discus hannai. In S. subcrenata, four POs were detected and their molecular masses were 391 kDa, 206 kDa, 174 kDa and <67 kDa, which were named as 391-PO, 206-PO, 174-PO and s-PO, respectively. Kinetic analyses indicated that all of the POs, except for 391-PO had higher affinity to L-DOPA and catechol than to hydroquinone and dopamine. However, all of the POs failed to oxidize tyrosine. The effects of divalent metal ions on POs’ activities were assayed, including Fe2+, Mg2+, Zn2+, Mn2+, Cu2+ and Ca2+ from FeCl2, MgSO4, ZnSO4, MnCl2, CuSO4 and CaCl2. The POs were inhibited by Fe2+ at all determined concentrations. Additionally, the inhibition assay showed that all of the POs were inhibited by cysteine, ascorbic acid, sodium sulfite, citric acid, ethylenediaminetetraacetic acid disodium (EDTA) and sodium diethyldithiocarbamate (DETC). The inhibition effects of critric acid and EDTA are dose-dependent. H. discus hannai PO and 391-PO were slightly inhibited by sodium azide, and H. discus hannai PO, 391-PO and 174-PO were slightly inhibited by thiourea. In conclusion, the POs in the three cultured mollusks are copper-containing laccase-type phenoloxidases with similar biochemical characteristics even though their molecular masses are different.

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Acknowledgements

This research was supported by the Qingdao National Laboratory for Marine Science and Technology (No. QNLM 2016ORP0307), the National Key Research and Development Program of China (No. YFD0900504), the National Basic Research Program of China (No. 2012CB114405), and the Taishan Scholar Program of Shandong Province.

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Authors and Affiliations

  1. Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003, China

    Xiangdi Song, Jingwei Jiang, Jing Xing & Wenbin Zhan

  2. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China

    Jing Xing & Wenbin Zhan

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  1. Xiangdi Song
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  2. Jingwei Jiang
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Correspondence to Jing Xing.

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Song, X., Jiang, J., Xing, J. et al. Isolation and Biochemical Characteristics Analyses of Phenoloxidases (POs) in Three Cultured Mollusk Species. J. Ocean Univ. China 21, 465–472 (2022). https://doi.org/10.1007/s11802-022-4808-6

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  • DOI: https://doi.org/10.1007/s11802-022-4808-6

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