日本蟳酚氧化酶的分离纯化及其部分生物化学性质研究

利用离子交换层析和凝胶过滤层析等方法,从日本(Charybdis japonica)血淋巴中分离纯化出了酚氧化酶,并以L-二羟苯丙氨酸(L-DOPA)作为特异性底物对其生化性质和酶性质进行了研究。结果表明,酚氧化酶和酚氧化酶原的分子质量分别为64.5 ku和69.5 ku。以L-DOPA为底物对酚氧化酶纯品进行研究发现,其最适pH值为6.0、最适温度为40℃。对底物L-DOPA和儿茶酚的米氏常数Km值分别为3.41和7.97 mmol/L。该酶对亚硫酸钠、苯硫脲极为敏感,对硫脲、苯甲酸非常敏感,表明该酶很可能是一种儿茶酚酶型的酶。此外,EDTA,DETC,Zn2 ,Mg2 和Cu2 均能显著抑制该酶活性,且10 mmol/L Cu2 能有效地回复该酶被DETC所抑制的酶活性,表明该酶确为一种Cu-金属酶。

Purification and partial characterization of phenoloxidase from crab Charybdis japonica

Phenoloxidase(PO) from hemolymph of Charybdis japonica was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties by using L-dihydroxyphenylalanine(L-DOPA) as the specific substrate in this study. It was found that prophenoloxidase(proPO), isolated as a monomeric protein, had a molecular weight of 69.5 ku, and a 64.5 ku PO molecule was often contained in preparations. The PO showed an optimal pH value of 6.0, and an optimal temperature of 40 °C, respectively. And an apparent K_m value of PO was 3.41 on L-DOPA, and 7.97 on catechol, respectively. PO activity was extremely sensitive to sodium sulfite and 1-phenyl-2-thiourea, and quite sensitive to thiourea and benzoic acid. Based on its sensitivity to different oxidase inhibitors and a higher affinity on catechol, this PO was thus classified as an o-diphenoloxidase. The PO activity was also strongly inhibited by Zn²⁺, Mg²⁺, thylenediaminetetraacetic acid(EDTA) and diethyldithiocarbamate(DETC). And the DETC-inhibited PO activity can be recovered perfectly by Cu²⁺. Then it can be concluded that Charybdis PO is most probably a metalloenzyme that required Cu²⁺ for its full activity.