A continuous fluorescence assay for phospholipase A2 with nontagged lipid.
Human nonpancreatic secreted phospholipase A2 (hnps PLA2) is considered to be an important drug target for antiinflammation therapy. We have established a new fluorescence assay by using 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial probe for hydrophobic environment detection. The fitted apparent k(cat)/K(m) of hnps PLA2 is 0.0181 +/- 0.0005 RFU/microMs. Tests on known synthesized inhibitor gave IC50 values similar to those from isotope-labeled assay. Because ANS is a commonly used probe for hydrophobic environment detection that needs no modification in the current assay, this strategy may be widely applicable for interfacial catalytic reactions.
Huang
Changkang
C
State Key Laboratory for Structural Chemistry of Stable and Unstable Species, College of Chemistry, Peking University, Beijing 100871, China.
Zhou
Lu
L
Liu
Ying
Y
Lai
Luhua
L
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D010741:Phospholipases A / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D000817:Anilino Naphthalenesulfonates; D008055:Lipids; C027132:1-anilino-8-naphthalenesulfonate; D010741:Phospholipases A; D054467:Phospholipases A2