Polyphenol oxidase from wheat bran is a serpin
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Polyphenol oxidase (PPO; EC 126.96.36.199) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The Km values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The Ki value for tropolone is 2.2 × 10-7 M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 × 10-7 M PPO. The Ki value for PPO is 2.3 × 10-8 M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.
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