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P2053
Novel Ratiometric Phospholipase A2 Specific Enzyme Substrate with a Fluorescence Donor & Acceptor Pair
Presenter Hee Chol Kang, Invitrogen Corporation, USA
Additional Authors: Laurie Cooley, Aimei Chen, Iain Johnson, Jerry Thomas, Nabi Malekzade,Kathy Free, Mike Ignatius, Bruce Branchaud,  and Hee Chol Kang
Phospholipase A2 (PLA2) is a term used to represent a superfamily of intracellular and secreted enzymes that hydrolyze the sn-2 ester linkage of phospholipids. PLA2 plays important roles in many human diseases, including cardiovascular, inflammatory, and nervous system disorders, and cancers. Even though many methods have been developed to monitor the activity of PLA2, there are limitations to each of those methods, including substrate specificity, background noise and their suitability for real-time continuous monitoring in living cells. We have designed and synthesized a novel PLA2 selective substrate, based on fluorescence resonance energy transfer, which emits a distinct fluorescent profile before and after cleavage by PLA2. BODIPY fluorophores were chosen as a donor and acceptor pair because of their lipid compatibility and of their high fluorescence QY and photostability. We have evaluated this novel substrate with phospholipases and demonstrated its advantages, e.g. high specificity for PLA2 and its fluorescence ratiometric capability and lower background. We will also discuss its potential use for quantitative real-time detection of PLA2  activity, and drug development assay, whether in cells, tissues and whole organisms.