Proton gradients are utilized by cells to power the transport activity of many membrane proteins. Synthetic cells, such as proteo-giant unilamellar vesicles, offer an advanced approach for studying the functionality of membrane proteins in isolation. However, understanding of protein-based transport in vitro requires accurate measurements of proton flux and its accompanying electrochemical gradient across the lipid bilayer. We present an approach to directly quantify the flux of protons across single cell-sized lipid vesicles under modulated electrochemical gradients. Our measurements reveal the corresponding association between proton permeation and transmembrane potential development and its relation to the chemical nature of the conjugated anion (base). In the case of formic acid, we showed that, out of the total amount of permeated protons, a fraction of ≈0.2 traverse the lipid bilayer as H⁺, with the rest (≈0.8) in the form of a neutral acid. For strong acids (HCl or HNO₃), proton permeation was governed by translocation of H⁺. Accordingly, a larger proton motive force (pmf) was generated for strong acids ($\text{pmf}=14.2$ mV) relative to formic acid ($\text{pmf}=1.3$ mV). We anticipate that our approach will guide the development of protein-based transport driven by proton gradient in artificial cell models and enable a deeper understanding of how vital acids, such as fatty acids, amino acids, bile acids, and carboxylic acid-containing drugs, traverse the lipid bilayer.

细胞利用质子梯度为许多膜蛋白的转运活动提供动力。合成细胞，例如蛋白质巨单层囊泡，为研究分离膜蛋白的功能提供了一种先进的方法。然而，了解体外基于蛋白质的运输需要准确测量质子通量及其伴随的跨脂质双层的电化学梯度。我们提出了一种在调制电化学梯度下直接量化穿过单细胞大小的脂质囊泡的质子通量的方法。我们的测量揭示了质子渗透和跨膜电位发展之间的相应关联及其与共轭阴离子（碱基）化学性质的关系。就甲酸而言，我们表明，在渗透质子的总量中，⁺，其余的（≈0.8）以中性酸的形式存在。对于强酸（HCl 或 HNO _{3 ），质子渗透由 H} ⁺的易位控制。因此，强酸会产生较大的质子动力（pmf）（$\text{脉冲频率}=14.2$mV) 相对于甲酸 ($\text{脉冲频率}=1.3$毫伏）。我们预计我们的方法将指导人工细胞模型中质子梯度驱动的基于蛋白质的运输的发展，并能够更深入地了解重要酸（例如脂肪酸、氨基酸、胆汁酸和含羧酸药物）如何穿过脂质双层。