A continuous fluorescence assay for phospholipase A2 with nontagged lipid

Changkang Huang; Lu Zhou; Ying Liu; Luhua Lai

doi:10.1016/j.ab.2006.01.021

A continuous fluorescence assay for phospholipase A2 with nontagged lipid

Anal Biochem. 2006 Apr 1;351(1):11-7. doi: 10.1016/j.ab.2006.01.021. Epub 2006 Feb 3.

Authors

Changkang Huang¹, Lu Zhou, Ying Liu, Luhua Lai

Affiliation

¹ State Key Laboratory for Structural Chemistry of Stable and Unstable Species, College of Chemistry, Peking University, Beijing 100871, China.

PMID: 16510113
DOI: 10.1016/j.ab.2006.01.021

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anilino Naphthalenesulfonates / chemistry
Humans
Kinetics
Lipids / chemistry*
Phospholipases A / analysis*
Phospholipases A / antagonists & inhibitors
Phospholipases A / chemistry
Phospholipases A2
Spectrometry, Fluorescence / methods*
Substrate Specificity

Substances

Anilino Naphthalenesulfonates
Lipids
1-anilino-8-naphthalenesulfonate
Phospholipases A
Phospholipases A2