Enzymatic Characterization and Functional Groups of Polyphenol Oxidase from the Pupae of Blowfly (Sarcophaga bullata)

Qin Wang¹, Qing-Xi Chen^1*, Xiao-Hong Huang², Li-Na Ke¹, Yan Shi¹, and Jun Wang¹

¹Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, China; fax: +86-5922185487; E-mail: chenqx@jingxian.xmu.edu.cn

²Biocide Research Center, Fujian Agriculture and Forestry University, Fuzhou 350002, China

^* To whom correspondence should be addressed.

Received January 8, 2004; Revision received March 6, 2004

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Polyphenol oxidase (EC 1.14.18.1) was purified from the pupae of blowfly (Sarcophaga bullata) by a procedure involving ammonium sulfate fractionation and chromatography on DEAE-cellulose and Sephadex G-100. Kinetic characteristics of the enzyme were determined using L-DOPA as substrate. The specific activity of the enzyme was 770 U/mg, and the Michaelis constant (K_m) was 1.5 ± 0.1 mM (pH 6.8, 30°C). Activity was maximal at 40°C, pH 6.5. Chemical modification experiments demonstrated that cysteine and tryptophan residues are essential and arginine residues are not essential to the enzyme function. The enzyme is inhibited by quercetin with an IC₅₀ of 0.20 ± 0.06 mM. The inhibition is of competitive type, and the inhibition constant was determined to be 88 µM.

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KEY WORDS: polyphenol oxidase, blowfly pupae, kinetic characterization, chemical modification

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