| Hemocyanins (Hcs), responsible for oxygen transport, are a kind of extracellular giant proteins found in the hemolymph of both mollusks and arthropods. Many studies indicate that although hemocyanins and phenoloxidases (PO) of mollusks and arthropods serve different physiological functions, but they both have copper binding sites, and Hc can be functionally converted into a kind of phenoloxidase-like enzyme (hemocyanin-derived phenoloxidase, HdPO) and display PO activities under certain conditions. Biochemically characterization of HdPO has not only great significance in understanding its roles in invertebrate non-specific immune system, but also helps us to fully cognize the multiple biological functions of Hc. In order to characterize the properties of HdPO biochemically, hemocyanin from hemolymph of Charybdis japonica was purified by sephacryl S-100 gel-filtration and DEAE sepharose Fast Flow ion-exchange chromatography, and HdPO was characterized biochemically by using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate in this study. It was found that the purified Hc, with an A340/A280 absorbance ratio of 0.21, had both 74.3 and 69.1 kDa molecules in SDS-PAGE (sodium dodecyl sulfate polyaerylemide gel electrophoresis), which are different from those of pro-phenoloxidase (proPO) and PO. Combined with the different eluting peaks of He and proPO-PO in column chromatography, it can be concluded that the protein purified in this study does be Charybdis He and has not been contaminated by proPO or PO. SDS, isopropanol, trypsin and urea were utilized to stimulate the convertion of Charybdis He into HdPO, and it was found that 0.1% SDS was the most effective stimulant, while isopropanol and trypsin v/ere less effective than SDS, but urea has almost no effect on HdPO convertion. Then, the purified Hc was stimulated with 0.1% SDS, and thegenerated HdPO was investigated biochemically and enzymatically by using L-DOPA as the specific substrate in this study. It was found that the optimum pH value and temperature of Charybdis HdPO was 7.0 and 40 ℃ respectively, and the apparent Km value of the HdPO was about 2.90 mmol/L on L-DOPA. Substrate specificity studies showed that this HdPO was capable of oxidizing not only Z--DOPA but also tyrosine, and with an apparent Km value of 7.33 mmol/L on tyrosine. However, this HdPO had no oxidizing activity on hydroquinone and catechol. From above in can be concluded that the HdPO had obvious oxidizing activity both on diphenol and monophenol, which implies that this HdPO was probably a type of tyrosinase-type PO but not a laccase type or catechol type. Besides, the HdPO was extremely sensitive to some specific inhibitors of tyrosinase such as cysteine, phenyl thiourea and ascorbic acid, very sensitive to thio urea and sodium sulfite, but not sensitive to benzoic acid and citric acid. Combined with its substrate specificity, it can be concluded that Charybdis HdPO is most probably a kind of tyrosinase-type PO. Moreover, the HdPO was also very sensitive to metal chelator and metal ions as well. Its PO-like enzyme activity can be greatly inhibited by Cu2+, thylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). Combined with the perfect recovering effect of 10 mmol/L Cu2+ on EDTA pre-inhibited HdPO activity, it can be concluded that Charybdis HdPO is a kind of Cu-metalloenzyme, the same as Charybdis PO. It also implies that Charybdis Hc is also a type of copper-containing protein, the same as Hc from Peneaus japonicus. This study makes us to understand more easily why Hc can be converted into HdPO in certain conditions, and plays the parallel immune function to PO in the invertebrate non-specific immune system. |
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