Optically Trapping Confocal Raman Microscopy of Individual Lipid Vesicles: Kinetics of Phospholipase A2-Catalyzed Hydrolysis of Phospholipids in the Membrane Bilayer
详细信息 查看全文
- 作者：Daniel P. Cherney ; Grant A. Myers ; Robert A. Horton ; Joel M. Harris
- 刊名：Analytical Chemistry
- 出版年：2006
- 出版时间：October 1, 2006
- 年：2006
- 卷：78
- 期：19
- 页码：6928 - 6935
- 全文大小：151K
- 年卷期：v.78,no.19(October 1, 2006)
- ISSN：1520-6882
文摘
Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2position of 1,2-dimyristoyl-sn-glycero-3-phosphocholinein optically trapped liposomes is monitored in situ usingconfocal Raman microscopy. Individual optically trappedliposomes (0.6 
m in diameter) are exposed to PLA2isolated from cobra (Naja naja naja) venom at varyingenzyme concentrations. The relative Raman scatteringintensities of C-C stretching vibrations from the trans andgauche conformers of the acyl chains are correlateddirectly with the extent of hydrolysis, allowing the progressof the reaction to be monitored in situ on a single vesicle.In dilute vesicle dispersions, the technique allows themuch higher local concentration of lipid molecules in asingle vesicle to be detected free of interferences from thesurrounding solution. Observing the local composition ofan optically trapped vesicle also allows one to determinewhether the products of enzyme-catalyzed hydrolysisremain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lagprior to the rapid hydrolysis. The lag time varied inverselywith the enzyme concentration, which is consistent withthe products of enzyme-catalyzed lipid hydrolysis reachinga critical concentration that allows the enzyme to react ata much faster rate. The turnover rate of membrane-boundenzyme determined by Raman microscopy during therapid, burst-phase kinetics was 1200 s-1. Based onprevious measurements of the equilibrium for PLA2binding to lipid membranes, the average number ofenzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quitesmall, only two PLA2 molecules at the lowest enzymeconcentration studied.
m in diameter) are exposed to PLA2isolated from cobra (Naja naja naja) venom at varyingenzyme concentrations. The relative Raman scatteringintensities of C-C stretching vibrations from the trans andgauche conformers of the acyl chains are correlateddirectly with the extent of hydrolysis, allowing the progressof the reaction to be monitored in situ on a single vesicle.In dilute vesicle dispersions, the technique allows themuch higher local concentration of lipid molecules in asingle vesicle to be detected free of interferences from thesurrounding solution. Observing the local composition ofan optically trapped vesicle also allows one to determinewhether the products of enzyme-catalyzed hydrolysisremain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lagprior to the rapid hydrolysis. The lag time varied inverselywith the enzyme concentration, which is consistent withthe products of enzyme-catalyzed lipid hydrolysis reachinga critical concentration that allows the enzyme to react ata much faster rate. The turnover rate of membrane-boundenzyme determined by Raman microscopy during therapid, burst-phase kinetics was 1200 s-1. Based onprevious measurements of the equilibrium for PLA2binding to lipid membranes, the average number ofenzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quitesmall, only two PLA2 molecules at the lowest enzymeconcentration studied.