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 PLA(2) 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 PLA(2) activity and assessment of the inhibitory effect of the non-steroidal anti-inflammatory drugs (NSAIDs) on PLA(2) activity. Several studies were performed with the aim of establishing the appropriate flow system configuration: the liposome substrate; PLA(2) 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 PLA(2) is effectively inhibited by the NSAIDs tested (meloxicam, tolmetin and ibuprofen) and by the alpha-lipoic acid, used as a positive control. Results 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.
Araujo
André R T S
AR
REQUIMTE, Serviço de Química-Física, Faculdade de Farmácia da Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal.
Gaspar
Diana
D
Lúcio
Marlene
M
Reis
Salette
S
Saraiva
M Lúcia M F S
ML
Lima
José L F C
JL
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001514:Bee Venoms / Q000201:enzymology; D017022:Flow Injection Analysis / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry; D006868:Hydrolysis; D008081:Liposomes / Q000378:metabolism*; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2 / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D008063:Thioctic Acid / Q000494:pharmacology; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001514:Bee Venoms; D005456:Fluorescent Dyes; D008081:Liposomes; D064801:Phospholipase A2 Inhibitors; C027132:1-anilino-8-naphthalenesulfonate; D008063:Thioctic Acid; D054467:Phospholipases A2