Purification and

Purification
and Partial Biochemical Characterization of Phenoloxidase from Penaeus
chinensis

FAN Ting-Jun^*, WANG Xiao-Feng
( Department of Marine Biology, College of Marine Life Sciences, Ocean
University of Qingdao, Qingdao 266003, China )

Abstract    Using L-DOPA
as a probe, phenoloxidase (PO) from Penaeus chinensis
hemolymph was purified by gel-filtration and ion-exchange chromatography, and
characterized in terms of its molecular weight and enzymatic properties in this
study. It was found that prophenoloxidase (proPO) isolated as monomeric protein
had a molecular weight of 87.5 kD, and a 77 kD phenoloxidase molecule was often
contained in preparations. The 87.5 kD proPO had a very low enzymatic activity
on 0.02 mmol/L L-DOPA, whereas the 77 kD PO had a very high
enzymatic activity on L-DOPA. Enzymatic activity of PO against
L-DOPA was optimal at pH 6.0, temperature of 40 ℃, and with an
apparent Km value of 1.99 mmol/L. The PO activity was extremely
sensitive to ascorbic acid, cysteine and dithiothreitol, very sensitive to thio
urea, however not sensitive to sodium sulfite, citric acid and benzoic acid,
confirmed that it was a phenoloxidase, combined with its specific oxidase
activity on substrate of monophenol and L-DOPA, indicate that
it was a tyrosinase type phenoloxidase. This enzyme is very sensitive to EDTA
and metal ions, its activity is strongly enhanced by Mg²⁺ and
strongly inhibited by Cu²⁺, which indicates that this PO is probably
a kind of metalloenzyme.
Key words  Penaeus chinensis; phenoloxidase; L-DOPA; tyrosinase;
metalloenzyme

^*Corresponding authors: Tel,
86-532-2032459; Fax,86-532-2032276; e-mail, [email protected]