Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama , 2630 Sugitani, Toyama 930-0194, Japan.
Langmuir. 2016 Dec 27;32(51):13697-13705. doi: 10.1021/acs.langmuir.6b03515. Epub 2016 Dec 12.
Methyl-β-cyclodextrin (MβCD) can transfer phospholipids between vesicles, and its transfer ability has been utilized for the preparation of asymmetric vesicle and lipid incorporation into culture cells. Nevertheless, a detailed kinetic analysis of the MβCD-mediated phospholipid transfer has not yet been carried out. We performed real-time monitoring of intervesicular lipid transfer by means of the fluorescence of pyrene-labeled phospholipids. Intermolecular excimer formation of the pyrene-labeled lipids in a membrane strongly depends on the local concentration of the fluorophore and decreases when the pyrene-labeled lipids are transferred from donor (fluorophore-containing) vesicles to acceptor (fluorophore-free) vesicles. We monitored the fluorescence intensity of the pyrene monomer and excimer simultaneously and found that the excimer/monomer ratio decreased in the presence of MβCD, pointing to MβCD-mediated lipid transfer. The transfer rate depended on the MβCD concentration but not on the lipid concentration, suggesting that dissociation from the membrane via extraction by MβCD is the rate-limiting step of the lipid transfer. Calibration of the excimer/monomer ratio to the molar fraction of the pyrene-labeled lipids enabled us to evaluate the dissociation rate constant correctly. From the temperature dependence of the transfer, we obtained the thermodynamic activation parameters, which revealed that the extraction of phosphatidylcholine by MβCD from membranes is less enthalpically unfavorable than that of phosphatidylglycerol and phosphatidylinositol.
甲基-β-环糊精（MβCD）可在囊泡之间转移磷脂，其转移能力已被用于制备不对称囊泡和将脂质掺入培养细胞。然而，MβCD 介导的磷脂转移的详细动力学分析尚未进行。我们通过使用芘标记的磷脂的荧光实时监测囊泡间脂质转移。膜中芘标记的脂质的分子间激基复合物形成强烈依赖于荧光团的局部浓度，并且当芘标记的脂质从供体（含荧光团）囊泡转移到受体（无荧光团）囊泡时，芘标记的脂质的激基复合物形成减少。我们同时监测芘单体和激基复合物的荧光强度，发现存在 MβCD 时激基复合物/单体比降低，表明 MβCD 介导的脂质转移。转移速率取决于 MβCD 浓度而不取决于脂质浓度，表明通过 MβCD 从膜中解吸是脂质转移的限速步骤。将激基复合物/单体比校准为芘标记的脂质的摩尔分数使我们能够正确评估解离速率常数。从转移的温度依赖性中，我们获得了热力学活化参数，这些参数表明 MβCD 从膜中提取磷脂酰胆碱的焓不利程度低于提取磷脂酰甘油和磷脂酰肌醇。
