The purpose of the study was to assess a fluorimetric assay for the determination of total phospholipase A(2) (PLA(2)) activity in biological samples introducing the innovation of immobilized substrates on crosslinked polymeric membranes. The immobilized C(12)-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 PLA(2). Atomic Force Microscopy revealed morphological alterations of the immobilized substrate after the reaction with PLA(2). Mass spectrometry showed that only C(12)-NBD-FA, the PLA(2 )hydrolysis product, was detected in the reaction mixture, indicating that PLA(2) recognizes PVA-SbQ/C(12)-NBD-PtdCho as a surface to perform catalysis.
Keywords: Fluorescence; HPLC; Mass spectrometry; Phospholipase A2; Phospholipases; Phospholipids.