Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting

Tsai Mu Cheng, Pei Chen Huang, Ju Pin Pan, Kuan Yu Lin, Simon J T Mao

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3 mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.

Original languageEnglish
Pages (from-to)331-336
Number of pages6
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume849
Issue number1-2
DOIs
Publication statusPublished - Apr 15 2007
Externally publishedYes

Fingerprint

Catechol Oxidase
Electrophoresis
Gels
Cucurbitaceae
Raphanus
Monophenol Monooxygenase
Agaricales
Vegetables
Enzyme Assays
Enzymes
Lycopersicon esculentum
Fruits
Sodium Dodecyl Sulfate
Detergents
Glass
Polyacrylamide Gel Electrophoresis
Assays
Fruit
Animals
Western Blotting

Keywords

  • Browning reaction
  • Catechol
  • Immobilized-paper
  • Natural PPO inhibitor
  • Polyphenol oxidase
  • Tyrosinase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting. / Cheng, Tsai Mu; Huang, Pei Chen; Pan, Ju Pin; Lin, Kuan Yu; Mao, Simon J T.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 849, No. 1-2, 15.04.2007, p. 331-336.

Research output: Contribution to journalArticle

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