Measurement of intravesicular volumes by salt entrapment

Internal volume is a very sensitive parameter of vesicle morphology. Measurement of captured volumes by solute entrapment is legitimate for most types of vesicles (Perkin, W.R. et al, (1993) Chem. Phys. Lipids 64, 197-217). In this study chloride was selected as the most convenient marker ion because the ubiquity of Cl- in physiological buffers eliminates prelabeling with exogeneous markers and because minute concentrations of trapped chloride are well detectable in the presence of large extravesicular nitrate concentrations. Perfect exchange of external chloride for nitrate was shown to be accomplished by gel filtration, dialysis, or sucrose gradient flotation but only after significant technical improvements and/or elimination of experimental pitfalls. Reliability was cross-checked by simultaneous entrapment of Cl- and K+. Diafiltration and ion exchange chromatography appeared inapplicable for exchange of extravesicular salt. When a representative variety of vesicle preparations was analyzed for internal volume (as well as for external surface and size) unexpected features of vesicle morphology were discovered. This emphasizes the genuine role of macroscopic vesicle characterization in complementing information from electron microscopy.