Molecular parameters of gangliosides in monolayers: comparative evaluation of suitable purification procedures.

The molecular area, collapse pressure, and surface potential of gangliosides obtained by different methods were systematically compared in monolayers at the air-water interface. Different values of these parameters are obtained depending on the purification procedure employed for the isolation of pure gangliosides. This is due to impurities (such as peptidaceous material) that remain in different amounts in the various preparations and that modify the ganglioside surface behavior. Routine purity checking by HPTLC analysis of gangliosides usually fails to reveal these impurities. On the other hand, even if the monolayer technique cannot identify the nature or amount of contaminants, it is extremely sensitive to reveal alterations of the surface molecular parameters caused by relatively small amounts of other components coextracted with the ganglioside or adventitiously introduced with the solvents or subphases employed. This is a serious problem for the obtention of correct and reproducible values of such important parameters as the molecular area of gangliosides, their electrostatic potential in oriented interfaces, and their interactions with other lipids and proteins. A procedure leading to consistent molecular parameters that remain reproducible after several repurification cycles is to perform an alkaline treatment on previously purified gangliosides species with NaOH, this is followed by dialysis against bidistilled water, rechromatography on DEAE-Sephadex A25, silicic acid or Iatrobeads, and Sephadex LH-20 columns; repurified gangliosides are stored in chloroform-methanol-0.01 M NaOH (60:30:4.5).