Sequential injection system for phospholipase A2 activity evaluation: Studies on liposomes using an environment-sensitive fluorescent probe

This work reports the development of an automatic methodology based on the use of 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial fluorescent probe for detecting the hydrophobic environment shift around the probe, caused by the hydrolytic action of PLA2 on the liposomes. The implementation of this reaction in a sequential injection analysis (SIA) system along with the use of the mixing chambers permitted the evaluation of PLA2 activity and assessment of the inhibitory effect of the non-steroidal anti-inflammatory drugs (NSAIDs) on PLA2 activity. l studies were performed with the aim of establishing the appropriate flow system configuration: the liposome substrate; PLA2 and ANS optimum concentrations and incubation times before and after the enzyme addition. Based on these studies, the optimum reaction conditions were selected. It was shown that PLA2 is effectively inhibited by the NSAIDs tested (meloxicam, tolmetin and ibuprofen) and by the α-lipoic acid, used as a positive control. s obtained from the flow system are in agreement with those provided by the comparison batch procedures. The proposed methodology is in fact more efficient and rapid than the comparison batch experiments, enabling the exact timing of fluidic manipulations and precise control of the reaction conditions.