Purification And Partial Characterization Of Phenoloxidase From Ruditapes Philippinarum

The defence system of invertebrates is dependent on the innate and non-specific defense mechanisms.It is great essential for invertebrates to improve their immune function by finding out the mechanism of non-specific immunity factors and cells.It is well known that phenoloxidase (PO), facilitates the synthesis of melanin by oxidation of monophenols to o-diphenols(monophenolase activity),as well as the oxidation of o-diphenols to corresponding o-quinones(o-diphenolase activity),then o-quinones form melanin, is a key component in the invertebrate non-specific immune system. There are many reports of the properties of POs in insects and arthropods, but little information is available on molluscan PO.Using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate, 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 molecule 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 Km value of the PO for L-DOPA was 2.2 mmol/L. The Km value of the PO for tyrosine was 6.0 mmol/L.It showed that L-DOPA had a higher affinity with PO than tyrosine. 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 PO from the Ruditapes philippinarum is probably a kind of tyrosinase-type phenoloxidase. The PO activity was strongly inhibited by thylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DETC), Zn2+, Ca2+ and Cu2+, as well as by Mg2+. The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu2+ on DETC-inhibited PO activity, indicate that PO from Ruditapes philippinarum is most probably a copper-containing metalloenzyme.