High-performance adsorption chromatography of proteins on 2-micron spherical beads of hydroxyapatite.

Chromatographic studies have been done with a 100 mm x 7.5 mm I.D. high-performance liquid chromatographic column of hydroxyapatite beads of diameters as small as 2 micron. The high resolving power was demonstrated by a gradient fractionation of nine model proteins and an isocratic fractionation into two immunologically active components of a highly purified toxic shock syndrome toxin-1 preparation which was homogeneous upon both ion-exchange and molecular sieve chromatography as well as isoelectric focusing. The resolution of the toxin preparation thus attests to the unique separation mechanism of hydroxyapatite. Characteristic of hydroxyapatite is that all proteins can be desorbed by phosphate ions but few are affected by chloride ions. gamma-Globulins are the only serum proteins which belong to the latter category. This property of gamma-globulin has been utilized in a simple procedure for its specific purification directly from whole serum; for the isolation of gamma-globulin on a large scale the hydroxyapatite can be replaced by brushite, CaHPO4.2H2O, which is simpler and cheaper to prepare. In a set of isocratic elution experiments with cytochrome c the peak width was determined at different flow-rates. As expected with macroporous beads, the peaks became much narrower as the flow-rate was lowered. For maximum resolution, low flow-rates must be used.