Abstract
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. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.