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 language | English |
|---|---|
| Pages (from-to) | 331-336 |
| Number of pages | 6 |
| Journal | Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences |
| Volume | 849 |
| Issue number | 1-2 |
| DOIs | |
| Publication status | Published - Apr 15 2007 |
| Externally published | Yes |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting
AU - Cheng, Tsai Mu
AU - Huang, Pei Chen
AU - Pan, Ju Pin
AU - Lin, Kuan Yu
AU - Mao, Simon J T
PY - 2007/4/15
Y1 - 2007/4/15
N2 - 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.
AB - 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.
KW - Browning reaction
KW - Catechol
KW - Immobilized-paper
KW - Natural PPO inhibitor
KW - Polyphenol oxidase
KW - Tyrosinase
UR - http://www.scopus.com/inward/record.url?scp=34047161583&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34047161583&partnerID=8YFLogxK
U2 - 10.1016/j.jchromb.2006.08.046
DO - 10.1016/j.jchromb.2006.08.046
M3 - Article
VL - 849
SP - 331
EP - 336
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
SN - 1570-0232
IS - 1-2
ER -