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Immobilization of lipid substrates: application on phospholipase A2 determination

Immobilization of lipid substrates: application on phospholipase A2 determination
Immobilization of lipid substrates: application on phospholipase A2 determination

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A2 (PLA2) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C12-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA2. Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA2. Mass spectrometry showed that only C12-NBD-FA, the PLA2 hydrolysis product, was detected in the reaction mixture, indicating that PLA2 recognizes PVA-SbQ/C12-NBD-PtdCho as a surface to perform catalysis.

Fluorescence, HPLC, Mass spectrometry, Phospholipase A, Phospholipases, Phospholipids
0024-4201
1259-1271
Karkabounas, Athanassios
3fdf8fac-b122-48d9-aea1-38c8ddec8d93
Georgiadou, Dimitra G.
84977176-3678-4fb3-a3dd-2044a49c853b
Argitis, Panagiotis
ab9c4ea6-3dd2-4e34-935d-81bfb360f358
Psycharis, Vassilios
fad102df-b322-4aa0-aaa8-2aeb847141e6
Nakos, George
eed98138-4243-4d30-aa0d-ba732092aaae
Kosmas, Agni M.
9fb40e11-4a11-4435-a4f1-667f666dfcd4
Lekka, Marilena E.
0557a414-c550-469a-8fb3-6707d30a83c1
Karkabounas, Athanassios
3fdf8fac-b122-48d9-aea1-38c8ddec8d93
Georgiadou, Dimitra G.
84977176-3678-4fb3-a3dd-2044a49c853b
Argitis, Panagiotis
ab9c4ea6-3dd2-4e34-935d-81bfb360f358
Psycharis, Vassilios
fad102df-b322-4aa0-aaa8-2aeb847141e6
Nakos, George
eed98138-4243-4d30-aa0d-ba732092aaae
Kosmas, Agni M.
9fb40e11-4a11-4435-a4f1-667f666dfcd4
Lekka, Marilena E.
0557a414-c550-469a-8fb3-6707d30a83c1

Karkabounas, Athanassios, Georgiadou, Dimitra G., Argitis, Panagiotis, Psycharis, Vassilios, Nakos, George, Kosmas, Agni M. and Lekka, Marilena E. (2015) Immobilization of lipid substrates: application on phospholipase A2 determination. Lipids, 50 (12), 1259-1271. (doi:10.1007/s11745-015-4076-y).

Record type: Article

Abstract

The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A2 (PLA2) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C12-NBD-PtdCho, a fluorescent analogue of phosphatidylcholine, exhibited excellent stability for 3 months at 4 °C and was not desorbed in the aqueous reaction mixture during analysis. The limit of detection was 0.5 pmol FA (0.2 pg) and the linear part of the response curve extended from 1 up to 190 nmol FA/h/mL sample. The intra- and inter-day relative standard deviations (%RSD), were ≤6 and ≤9 %, respectively. Statistical comparison with other fluorescent methods showed excellent correlation and agreement. Semiempirical calculations showed a fair amount of electrostatic interaction between the NBD-labeled substrate and the crosslinked polyvinyl alcohol with the styryl pyridinium residues (PVA-SbQ) material, from the plane of which, the sn-2 acyl chain of the phospholipid stands out and is accessible by PLA2. Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA2. Mass spectrometry showed that only C12-NBD-FA, the PLA2 hydrolysis product, was detected in the reaction mixture, indicating that PLA2 recognizes PVA-SbQ/C12-NBD-PtdCho as a surface to perform catalysis.

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More information

e-pub ahead of print date: 8 October 2015
Published date: 1 December 2015
Keywords: Fluorescence, HPLC, Mass spectrometry, Phospholipase A, Phospholipases, Phospholipids

Identifiers

Local EPrints ID: 439827
URI: http://eprints.soton.ac.uk/id/eprint/439827
ISSN: 0024-4201
PURE UUID: 08dae41e-d424-4d36-b320-f0d6a0b5293d
ORCID for Dimitra G. Georgiadou: ORCID iD orcid.org/0000-0002-2620-3346

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Date deposited: 05 May 2020 16:30
Last modified: 07 Oct 2020 02:27

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Contributors

Author: Athanassios Karkabounas
Author: Panagiotis Argitis
Author: Vassilios Psycharis
Author: George Nakos
Author: Agni M. Kosmas
Author: Marilena E. Lekka

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