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Plant Physiol. Nov 1973; 52(5): 501–507.
PMCID: PMC366532

Purification and Some Properties of Two Polyphenol Oxidases from Bartlett Pears 1


Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with Km values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. Vmax and Km values for oxygen and chlorogenic acid were 103 μmoles O2 uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. Km values for chlorogenic acid were independent of pH from 3 to 7; the Vmax values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with Ki values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with Ki values of 0.04 and 0.11 mm for enzymes A and B, respectively.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • CLAYTON RA. Properties of tobacco polyphenol oxidase. Arch Biochem Biophys. 1959 Apr;81(2):404–417. [PubMed]
  • Duckworth HW, Coleman JE. Physicochemical and kinetic properties of mushroom tyrosinase. J Biol Chem. 1970 Apr 10;245(7):1613–1625. [PubMed]
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  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
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  • SIEGELMAN HW. Detection and identification of polyphenoloxidase substrates in apple and pear skins. Arch Biochem Biophys. 1955 May;56(1):97–102. [PubMed]
  • Vaughan PF, Butt VS. The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.). Biochem J. 1970 Aug;119(1):89–94. [PMC free article] [PubMed]
  • Wong TC, Luh BS, Whitaker JR. Isolation and characterization of polyphenol oxidase isozymes of clingstone peach. Plant Physiol. 1971 Jul;48(1):19–23. [PMC free article] [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists


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