Next Article in Journal
Total Synthesis and Metabolic Stability of Hispidulin and Its d-Labelled Derivative
Next Article in Special Issue
Ferulaldehyde Improves the Effect of Methotrexate in Experimental Arthritis
Previous Article in Journal
Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)2 and Its Serum Degradation Product against Multidrug-Resistant Bacteria
Previous Article in Special Issue
A Rapid and Simple TLC-Densitometric Method for Assay of Clobetasol Propionate in Topical Solution
Article Menu

Export Article

Open AccessArticle
Molecules 2017, 22(11), 1899; doi:10.3390/molecules22111899

HPLC Analysis and Biochemical Characterization of LOX from Eschscholtzia californica Cham.

1
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University in Bratislava, Kalinčiakova 8, 832 32 Bratislava, Slovak Republic
2
Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, 832 32 Bratislava, Slovak Republic
3
Toxicological and Antidoping Center, Faculty of Pharmacy, Comenius University in Bratislava, Odbojárov 10, 832 32 Bratislava, Slovak Republic
*
Author to whom correspondence should be addressed.
Received: 28 September 2017 / Revised: 31 October 2017 / Accepted: 31 October 2017 / Published: 4 November 2017
View Full-Text   |   Download PDF [1804 KB, uploaded 4 November 2017]   |  

Abstract

Background: Plant lipoxygenases (LOXs, EC 1.13.11.12) are involved in lipid degradation, regulation of growth and development, senescence, and defence reactions. LOX represents the starting enzyme of the octadecanoid pathway. The aim of the work was to purify LOX from California poppy (Eschscholtzia californica Cham.), to determine its biochemical properties and to identify and quantify the products of LOX reaction with unsaturated fatty acids. Methods: LOX from California poppy seedlings was purified by hydrophobic chromatography (Phenyl-Sepharose CL-4B) and by ion-exchange chromatography (Q-Sepharose). The isolated LOX was incubated with linoleic acid used as a substrate. The HPLC experiments were performed with the Agilent Technologies 1050 series HPLC system. For the preparative separation of a mixture of hydroxy fatty acids from the sample matrix, the RP-HPLC method was used (column 120-5 Nucleosil C18). Then, the NP-HPLC analysis (separation, identification, and determination) of hydroxy fatty acid isomers was carried out on a Zorbax Rx-SIL column. Results: The purified LOX indicates the presence of a nontraditional plant enzyme with dual positional specificity (a ratio of 9- and 13-hydroperoxide products 1:1), a relative molecular mass of 85 kDa, a pH optimum of 6.5, an increasing activity stimulation by CaCl2 till 2 mM, and a high substrate reactivity to linoleic acid with kinetic values of KM 2.6 mM and Vmax 3.14 μM/min/mg. Conclusions: For the first time, the LOX from California poppy seedlings was partially purified and the biochemical properties of the enzyme were analyzed. A dual positional specificity of the LOX found from California poppy seedlings is in agreement with the results obtained for LOXs isolated from other Papaveraceaes. A 1:1 ratio of 9-/13-HODE is attractive for the simultaneous investigation of both biotic stress responses (indicated by the 9-HODE marker) and the biosynthesis of jasmonic acid and jasmonates (indicated by the 13-HODE marker). View Full-Text
Keywords: lipoxygenase; hydroxy fatty acid isomers; Eschscholtzia californica Cham.; purification; HPLC analysis; biochemical parameters lipoxygenase; hydroxy fatty acid isomers; Eschscholtzia californica Cham.; purification; HPLC analysis; biochemical parameters
Figures

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. (CC BY 4.0).

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Kollárová, R.; Holková, I.; Rauová, D.; Bálintová, B.; Mikuš, P.; Obložinský, M. HPLC Analysis and Biochemical Characterization of LOX from Eschscholtzia californica Cham.. Molecules 2017, 22, 1899.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]

Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top