Optically trapping confocal Raman microscopy of individual lipid vesicles: kinetics of phospholipase A(2)-catalyzed hydrolysis of phospholipids in the membrane bilayer.
Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 microm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C-C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s(-1). Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied.
Cherney
Daniel P
DP
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, USA.
Myers
Grant A
GA
Horton
Robert A
RA
Harris
Joel M
JM
D002384:Catalysis; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers; D008055:Lipids / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D013059:Spectrum Analysis, Raman / Q000379:methods*
D008051:Lipid Bilayers; D008055:Lipids; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2