Stopped-flow Spectrophotometry to Measure Water Permeability

The setup of stopped-flow spectrophotometry was done as per a previous report [12 (link)]. In brief, liposomes or proteo-liposomes were measured at 436 nm in a stopped-flow spectrophotometer (SFM 300, Bio-Logic SAS, Claix, France). Sample suspensions were quickly mixed with equal volumes of hyperosmotic solution (assay buffer A with 400 mM sucrose). Data obtained from the spectrophotometer was fitted into an exponential rise equation; the initial shrinkage rate (k) was determined by the fitted curve of 6–10 measurements. The water permeability factor (P_f) of the vesicle samples was calculated using the equation described previously [9 (link),12 (link),19 (link)]: $P_f=\frac{k}{(S/V_0)\times V_w\times {∆}_{osm}}$
where S/V₀ is the vesicle’s initial surface-to-volume ratio, V_w is the partial molar volume of water (18 cm³/mol), and ${∆}_{osm}$ is the osmotic driving force. The S/V₀ was calculated by determining the diameters of the proteo-liposomes using dynamic light scattering (ZetaPlus particle sizing software 2.27). The ${∆}_{osm}$ was 200 mM in this case.