Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.)
1
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Kalinčiakova 8, 832 32 Bratislava, Slovakia
2
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, 832 32 Bratislava, Slovakia
3
Toxicological and Antidoping Center, Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, 832 32 Bratislava, Slovakia
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(23), 4268; https://doi.org/10.3390/molecules24234268
Received: 3 October 2019
/
Revised: 20 November 2019
/
Accepted: 21 November 2019
/
Published: 23 November 2019
(This article belongs to the Section Natural Products Chemistry)
Opium poppy (Papaver somniferum L.) is an ancient medicinal plant producing pharmaceutically important benzylisoquinoline alkaloids. In the present work we focused on the study of enzyme lipoxygenase (LOX, EC 1.13.11.12) from opium poppy cultures. LOX is involved in lipid peroxidation and lipoxygenase oxidation products of polyunsaturated fatty acids have a significant role in regulation of growth, development and plant defense responses to biotic or abiotic stress. The purpose of this study was to isolate and characterize LOX enzyme from opium poppy callus cultures. LOX was purified by ammonium sulfate precipitation and then followed by hydrophobic chromatography using Phenyl-Sepharose CL-4B and hydroxyapatite chromatography using HA Ultrogel sorbent. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting revealed that LOX from opium poppy cultures was a single monomeric protein showing the relative molecular weight of 83 kDa. To investigate the positional specificity of the LOX reaction, purified LOX was incubated with linoleic acid and the products were analyzed by high-performance liquid chromatography in two steps, firstly with reverse phase (120-5 Nucleosil C18 column) and secondly with normal phase (Zorbax Rx-SIL column). LOX converted linoleic acid primarily to 13-hydroperoxy-(9Z,11E)-octadecadienoic acids (78%) and to a lesser extent 9-hydroperoxy-(10E,12Z)-octadecadienoic acids (22%). Characterization of LOX from opium poppy cultures provided valuable information in understanding LOX involvement in regulation of signaling pathways leading to biosynthesis of secondary metabolites with significant biological activity.
View Full-Text
Keywords:
Papaver somniferum L.; lipoxygenase; purification; lipoxygenase products; positional specificity; HPLC analysis
▼
Show Figures
Graphical abstract
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
MDPI and ACS Style
Holková, I.; Rauová, D.; Mergová, M.; Bezáková, L.; Mikuš, P. Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.). Molecules 2019, 24, 4268. https://doi.org/10.3390/molecules24234268
AMA Style
Holková I, Rauová D, Mergová M, Bezáková L, Mikuš P. Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.). Molecules. 2019; 24(23):4268. https://doi.org/10.3390/molecules24234268
Chicago/Turabian StyleHolková, Ivana, Drahomíra Rauová, Michaela Mergová, Lýdia Bezáková, and Peter Mikuš. 2019. "Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.)" Molecules 24, no. 23: 4268. https://doi.org/10.3390/molecules24234268
Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.
