thiourea and sodium-sulfite

Phenoloxidase (PO) from hemolymph of Charybdis japonica was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. It was found that prophenoloxidase (proPO), isolated as a monomeric protein, had a molecular mass of 69.5 kDa, and a 64.5 kDa PO molecule was often contained in preparations. The PO activity showed optimal pH of 6.0, optimal temperature of 40 degrees C, and an apparent Km value of 3.41 on L-DOPA, and 7.97 on catechol. PO activity was extremely sensitive to sodium sulfite and 1-phenyl-2-thiourea, and very sensitive to thiourea and benzoic acid. Based on its inhibition characteristics and the substrate affinity, this PO was classified as a kind of o-diphenoloxidase. The PO activity was also strongly inhibited by Zn(2+), Mg(2+), ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). 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 Charybdis PO is most probably a copper-containing metalloenzyme.

Topics: Animals; Benzoic Acid; Brachyura; Chromatography; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Kinetics; Levodopa; Metals, Heavy; Monophenol Monooxygenase; Substrate Specificity; Sulfites; Thiocarbamates; Thiourea