High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of secretory phospholipase A(2).
A non-radioactive spectrometric assay for the evaluation of inhibitors of pancreatic group IB and non-pancreatic group IIA secretory phospholipase A(2) (sPLA(2)) is described. Mixed-micelles consisting of 1 mM of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol and 6 mM of sodium deoxycholate were used as substrate. The enzyme activity was determined directly without any sample clean-up by measuring the sPLA(2)-mediated oleic acid release with reversed-phase HPLC and UV-detection at 200 nm. The known sPLA(2) inhibitors MJ33 and AR-C 67047MI were analyzed in this assay for their inhibitory potency. While MJ33 revealed only a very weak inhibition of group IB and IIA sPLA(2) at the highest test concentration (33 microM), AR-C 67047MI proved to be a potent inhibitor of both enzymes with IC(50)-values of 0.36 and 0.14 microM, respectively.
Schmitt
Melanie
M
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, D-48149, Münster, Germany.
Lehr
Matthias
M
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology*; D006801:Humans; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D004791:Enzyme Inhibitors; D010741:Phospholipases A