Fluorescence Single‐Molecule Study of Cobra Phospholipase A 2 Action on a Supported Gel‐Phase Lipid Bilayer

Enzyme in action : The accumulated single‐molecule image (see picture) of phospholipase A 2 (PLA 2 ) 15–21 min after its injection on a lipid membrane shows transient interactions of PLA 2 (blue) with the membrane surface and stable binding to the boundaries of membrane structure defects. The adsorbed PLA 2 molecules move with a speed below the experimental resolution of 40 nm min −1 .Previous nanoscale investigations of the gel‐state membrane surface structure under the action of phospholipase A 2 (PLA 2 ) suggest that single enzymes at work scoot on the membrane surface from the observed defects, which creates nanosized channels oriented along the lipid crystal‐packing structure. To date, however, there have been no reports of direct observation of PLA 2 at the single‐molecule level focusing on how the enzymes interact with the defects. Herein, we report a single‐molecule fluorescence microscopy study on the action of enzymatically active rhodamine B‐labeled cobra PLA 2 on a supported lipid membrane with visible packing defects on a glass substrate. Working with a gel‐state phospholipid bilayer, the low‐activity period (lag phase) of PLA 2 action is followed by the burst binding of PLA 2 molecules from aqueous solution on a few newly created active sites. These active sites are distinguished by a spatial resolution of ≈40 nm, which is below the diffraction limit. The rate of active‐site propagation as reflected by new PLA 2 binding on the membrane surface is estimated to be ≈5 nm min −1 . This rate is about two orders of magnitude slower than the propagation rate of hydrolyzed channels estimated by AFM studies on bee venom PLA 2 on a similar membrane surface. This direct observation of PLA 2 molecules allows the visualization of different PLA 2 binding modes on the membrane surface and on the membrane boundary.