Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario, Canada.
Institute for Pharmaceutical Sciences, University of Freiburg , Freiburg, Germany.
Langmuir. 2016 Nov 8;32(44):11655-11663. doi: 10.1021/acs.langmuir.6b03292. Epub 2016 Oct 19.
Interactions between detergents and model membranes are well described by the three-stage model: saturation and solubilization boundaries divide bilayer-only, bilayer-micelle coexistence, and micelle-only ranges. An underlying assumption of the model is the equilibration of detergent between the two membrane leaflets. However, many detergents partition asymmetrically at room temperature due to slow flip-flop, such as sodium dodecyl sulfate (SDS) and lysolipids. In this work, we use isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) to investigate the solubilization of unilamellar POPC vesicles by 12:0 lysophosphocholine (12:0 LPC). Flip-flop of 12:0 LPC occurs beyond the time scale of our experiments, which establish a characteristic nonequilibrated state with asymmetric distribution: 12:0 LPC partitions primarily into the outer leaflet. Increasing asymmetry stress in the membrane does not lead to membrane failure, i.e., "cracking in" as seen for alkyl maltosides and other surfactants; instead, it reduces further membrane insertion which leads to the "staying out" of 12:0 LPC in solution. At above the critical micellar concentration of 12:0 LPC in the presence of the membrane, micelles persist and accommodate further LPC but take up lipid from vesicles only very slowly. Ultimately, solubilization proceeds via the micellar mechanism (Kragh-Hansen et al., 1995). With a combination of demicellization and solubilization experiments, we quantify the molar ratio partition coefficient (0.6 ± 0.1 mM) and enthalpy of partitioning (6.1 ± 0.3 kJ·mol) and estimate the maximum detergent/lipid ratio reached in the outer leaflet (<0.13). Despite the inapplicability of the three-stage model to 12:0 LPC at room temperature, we are able to extract quantitative information from ITC solubilization experiments and DLS that are important for the understanding of asymmetry-dependent processes such as endocytosis and the gating of mechanosensitive channels in vitro.
饱和和溶解边界将双层膜、双层-胶束共存和胶束仅存在的范围分开。该模型的一个基本假设是两亲分子在两个膜叶之间达到平衡。然而，许多两亲分子由于翻转缓慢而在室温下不对称分配，如十二烷基硫酸钠（SDS）和溶血磷脂。在这项工作中，我们使用等温滴定量热法（ITC）和动态光散射（DLS）研究了 12:0 溶血磷脂酰胆碱（12:0 LPC）对单层 POPC 囊泡的增溶作用。12:0 LPC 的翻转发生在我们实验的时间尺度之外，这建立了一个具有不对称分布的特征非平衡状态：12:0 LPC 主要分配到外层。增加膜的不对称性压力不会导致膜破裂，即不会像烷基麦芽糖和其他表面活性剂那样出现“内裂”；相反，它会减少进一步的膜插入，从而导致 12:0 LPC 在溶液中“留在外面”。在存在膜的情况下，当 12:0 LPC 的临界胶束浓度以上时，胶束仍然存在并容纳更多的 LPC，但非常缓慢地从囊泡中摄取脂质。最终，增溶通过胶束机制进行（Kragh-Hansen 等人，1995 年）。通过去胶束化和增溶实验的结合，我们定量了摩尔比分配系数（0.6±0.1 mM）和分配焓（6.1±0.3 kJ·mol），并估计了外层达到的最大表面活性剂/脂质比（<0.13）。尽管三阶段模型在室温下不适用于 12:0 LPC，但我们能够从 ITC 增溶实验和 DLS 中提取出对理解不对称依赖过程（如内吞作用和体外机械敏感通道的门控）很重要的定量信息。
