Abstract

We have analyzed the purification of charged giant unilamellar vesicles (GUVs) prepared in a buffer containing various concentrations of salt using their size distribution. The membranes of GUVs were synthesized by a mixture of dioleoylphosphocholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) lipids. The DOPG mole fractions (X) in the membranes of GUVs were 0.10, 0.25, 0.40, 0.55, 0.70, 0.90 in a physiological buffer containing 162 mM salt. In addition, for a fixed value of X the concentrations of salt (C) in the buffer were 12, 62, 112, 162, 212, 312, 362 mM. The size distribution histograms of experimentally investigated unpurified and purified GUVs were fitted with the lognormal distribution and obtained the multiplication factor \({\gamma }\) for mean (\({\mu }\)) and \({\eta }\) for standard deviation (\({\sigma }\)) of the lognormal distribution. The key parameters \(\gamma \) and \(\eta \) were responsible for changing the average size and size distribution of unpurified GUVs to purified ones. The theoretically fitting equation of experimentally obtained X- and C-dependent values of \(\gamma \) and \(\eta \) provided the calibration equation for estimating the average size of purified GUVs theoretically for any values of X and C. The estimated size of purified GUVs increased with the increase in electrostatic effect (i.e., increase in vesicle surface charge density or decrease in salt concentration in buffer). The estimated size of purified GUVs varied with X and C, which supported the previous report qualitatively. These investigations might be helpful in the field of cell/chemical biology for understanding the process of purification of vesicles/cells investigated by any other techniques.

Graphic Abstract

中文翻译：

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使用大小分布分析缓冲液中带电大泡的纯化

摘要

我们已经分析了使用大小分布在包含各种浓度盐的缓冲液中制备的带电巨型单层囊泡（GUV）的纯化。GUVs的膜是由二油酰基磷脂酰胆碱（DOPC）和二油酰基磷脂酰甘油（DOPG）脂质的混合物合成的。在含有162 mM盐的生理缓冲液中，GUV膜中的DOPG摩尔分数（X）为0.10、0.25、0.40、0.55、0.70、0.90。另外，对于X的固定值，缓冲液中盐（C）的浓度为12、62、112、162、212、312、362 mM。实验研究的未纯化和纯化的GUV的尺寸分布直方图与对数正态分布拟合，并获得了乘法因子\（{\ gamma} \）表示对数正态分布的平均值（\（{\ mu} \））和\（{\ eta} \）表示标准偏差（\（{\ sigma} \））。关键参数\（\ gamma \）和\（\ eta \）负责将未纯化的GUV的平均尺寸和尺寸分布更改为已纯化的GUV。实验获得的与X和C有关的\（\ gamma \）和\（\ eta \）值的理论拟合方程提供了校准方程，可用于从理论上估计任意X和C值的纯化GUV的平均尺寸。纯化的GUV的估计大小随静电效应的增加而增加（即，囊泡表面电荷密度的增加或缓冲液中盐浓度的减少）。纯化的GUV的估计大小随X和C的变化而变化，这在质量上支持了先前的报告。这些研究在细胞/化学生物学领域中有助于理解通过任何其他技术研究的囊泡/细胞的纯化过程。

图形摘要