Transmembrane pH Gradients Afforded Autocatalytically by a Lumenal Formaldehyde-Sulfite Clock Reaction

<jats:p> Proton gradients across membranes are central to modern bioenergetics and are thought to have played a pivotal role in life’s emergence. We report that the autocatalytic formaldehyde-sulfite (FS) pH-clock reaction can generate such gradients when encapsulated in unilamellar phospholipid vesicles. POPC/POPG vesicles (~100 nm) containing sodium (bi)sulfite and the fluorescent pH probe HPTS were prepared by the thin-film hydration method, extruded and purified via size exclusion chromatography. Upon aqueous formaldehyde addition, a rapid rise in lumen pH was observed, resulting in a transmembrane pH gradient whose magnitude and kinetics were sensitive to initial lumen pH. Vesicles with lumen pH near 7.5 exhibited an increase to ~8.8 followed by a gradual decrease toward neutrality. <jats:sup>13</jats:sup> C NMR spectroscopy confirmed encapsulation of the final FS product, hydroxymethanesulfonate. Vesicles doped with small percentages of oleic acid afforded faster proton transport, demonstrating the spike in pH can be controlled through membrane composition. These findings establish a simple, nonenzymatic route to transmembrane pH gradients and provide a rare experimental example of an autocatalytic reaction network confined within protocell-like vesicles. </jats:p>