Purification and Product Characterization of Lipoxygenase from Opium Poppy Cultures (Papaver somniferum L.)

Autor: Michaela Mergová, Ivana Holková, Lýdia Bezáková, Drahomíra Rauová, Peter Mikuš
Rok vydání: 2019
Předmět:
0106 biological sciences
chemistry_other
purification
HPLC analysis
Linoleic acid
Secondary Metabolism
Pharmaceutical Science
Opium Poppy
01 natural sciences
High-performance liquid chromatography
Article
positional specificity
Analytical Chemistry
Linoleic Acid
03 medical and health sciences
Lipoxygenase
chemistry.chemical_compound
Biosynthesis
Papaver somniferum L
Drug Discovery
Chemical Precipitation
Papaver
Physical and Theoretical Chemistry
Benzylisoquinoline
Chromatography
High Pressure Liquid
Ammonium sulfate precipitation
Plant Proteins
030304 developmental biology
0303 health sciences
integumentary system
biology
Chemistry
Sepharose
Organic Chemistry
food and beverages
biology.organism_classification
lipoxygenase
Molecular Weight
Durapatite
Biochemistry
Chemistry (miscellaneous)
biology.protein
Molecular Medicine
Lipid Peroxidation
lipoxygenase products
010606 plant biology & botany
Zdroj: Molecules
Volume 24
Issue 23
ISSN: 1420-3049
DOI: 10.3390/molecules24234268
Popis: 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.
Databáze: OpenAIRE
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