Chromatography of acidic phospholipids on immobilized neomycin

Columns of immobilized neomycin (reductively coupled to porous glass beads) were used to separate weakly acidic lipids as well as the polyphosphoinositides. All anionic lipids present in chloroform-methanol extracts, which had been washed first with acid and then with neutral salt solutions, were adsorbed. Phosphatidylserine and phosphatidic acid were eluted with chloroform-methanol-formic acid mixtures. Phosphatidylinositol and cardiolipin were eluted sequentially by very low concentrations (10-100 mM) of ammonium formate in chloroform-methanol-water. All three phosphoinositides were isolated from washed chloroform-methanol-KCl extracts of brain. Sulfatides eluted with the phosphatidylinositol. Phosphatidylinositol phosphate and phosphatidylinositol bisphosphate were eluted in turn by higher salt concentrations (0.4-1 M). The immobilized neomycin was used repeatedly but the capacity eventually declined. This loss was reversed by sodium borohydride reduction.