Hydroxylapatite Chromatography of Protein-Sodium Dodecyl Sulfate Complexes

Abstract

Abstract Hydroxylapatite (calcium phosphate) chromatography was investigated as a method for separating protein subunits dissociated with sodium dodecyl sulfate (SDS). The proteins used included: a series of 11 well characterized proteins composed of single or identical subunits varying in molecular weight from 11,700 to 165,000; hemoglobin, composed of α and β chains of 146 and 141 amino acids; and vaccinia virus structural proteins, composed of more than 15 different polypeptide subunits. Reduced proteins were complexed with SDS and adsorbed to columns of hydroxylapatite. Using linear gradients of sodium phosphate, pH 6.4, containing 0.1% SDS and 1mm dithiothreitol, all tested proteins eluted between 0.2 and 0.5 m phosphate. A useful feature of the method is that proteins do not all elute in order of molecular weight. Thus polypeptide separations on hydroxylapatite are different from those obtained by polyacrylamide gel electrophoresis or gel filtration in SDS. The high resolution of the method was demonstrated by separating the α and β chains of hemoglobin and complex mixtures of viral polypeptides. By using polyacrylamide gel electrophoresis and hydroxylapatite chromatography in succession, it has been possible to separate vaccinia virus structural polypeptides that had not previously been resolved.