Purification and characterization of phenoloxidase from clam Ruditapes philippinarum

• Published in: Fish & shellfish immunology 2005, Vol.18 (1), p.61-70
• Main Authors: Cong, Rishan, Sun, Wenjie, Liu, Guangxing, Fan, Tingjun, Meng, Xianghong, Yang, Lingling, Zhu, Liyan
• Format: Article
• Language: English
• Subjects: Animals; Bivalvia - enzymology; Chromatography, Gel; Chromatography, Ion Exchange; Ditiocarb - metabolism; Edetic Acid - metabolism; Electrophoresis, Polyacrylamide Gel; Hemolymph - metabolism; Kinetics; l-Dihydroxyphenylalanine; Levodopa; Metalloenzyme; Metals, Heavy - metabolism; Monophenol Monooxygenase - blood; Monophenol Monooxygenase - chemistry; Monophenol Monooxygenase - isolation & purification; Phenoloxidase; Ruditapes philippinarum; Tyrosinase
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2004.06.001

Using l-dihydroxyphenylalanine ( l-DOPA) as a specific substrate, phenoloxidase (PO) from clam ( Ruditapes philippinarum) was purified by Q Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-100 gel-filtration, and characterized biochemically and enzymatically in this study. The molecular mass of PO in SDS-PAGE is about 76.9 kDa, and the prophenoloxidase (proPO) molecule, isolated as a monomeric protein, is 84.1 kDa. The PO molecule had a high oxidative activity, and the proPO molecule had almost no oxidative activity. The PO activity was optimal at pH 7.0 and temperature of 40 °C. The K m value of the PO for l-DOPA was 2.2 mmol l −1. The PO was extremely sensitive to benzoic acid and sodium sulfite, very sensitive to citric acid, thio urea, 1-phenyl-2-thiourea and cysteine, but not sensitive to ascorbic acid. Combined with its specific enzyme activity on tyrosine and l-DOPA, it can be concluded that the Ruditapes PO is probably a kind of tyrosinase-type phenoloxidase. The PO activity was strongly inhibited by ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DETC), Zn 2+, Ca 2+ and Cu 2+, as well as by Mg 2+. The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu 2+ on DETC-inhibited PO activity, indicate that Ruditapes PO is most probably a copper-containing metalloenzyme.