New experimental approaches to the isoelectric fractionation of cells

Several parameters that could affect the survival rate and the resolution of cells in continuous-flow isoelectric focusing have been investigated. Cell survival at progressively lower pHs was found to be a function of the osmolarity of the medium: at pH 4 there was an absolute requirement for a 300 mOsm environment. A pH-dependent interaction of the carrier ampholytes with the cell surface has been demonstrated. The binding was very strong at pH 4, weak at pH 5 and totally abolished above pH 5. The same interaction was obtained with pentaethylenehexamine, the polyamino backbone of carrier ampholytes, stripped of carboxyl groups. A model was derived showing how at pH 4 the oligocationic carrier ampholytes, via their nitrogen groups, would bind to the polyanionic cell surface. The Hannig apparatus was adapted for cell separation in a continuously flowing curtain of isoelectric ampholytes. The medium osmolarity was maintained with glycine (delta pK 7.2), taurine (delta pK 8.5) and trimethylaminopropionyl sulphonate (TMAPS, delta pK 11). With increasing delta pK, the pH range that can be created in the presence of these compounds progressively widens, from a pH 4.3-7.5 range with glycine, to pH 3.5-7.5 with taurine up to pH 3.5-9.5 with TMAPS. The 48 fractions collected were routinely assayed for conductivity, osmolarity and pH.