Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method

Abstract

A method was developed which enables the characterization and fractionation of chromosomal proteins according to their chromatin binding properties. The method is based on the ability of hydroxyapatite to bind native chromatin in solutions which do not dissocciate chromosomal proteins from the DNA. The proteins are then selectively dissociated from the immobilized chromatin by treatment with NaCl, urea, or guanidine HCl. The hydroxyapatite dissociation method represents a rapid one-step fractionation procedure which results in the quantitative recovery of chromosomal proteins devoid of nucleic acids and is suitable for large preparations. The hydroxyapatite dissociation method provides a versatile procedure for the study and preparation of chromosomal proteins. The patterns of dissociation of both histones and nonhistone chromosomal proteins by NaCl and urea from chicken oviduct chromatin were characterized by this method. In addition, this technique enabled the purification of the major histone species in a single operation. Partial purification of specific nonhistone proteins, including the estrogen receptor, was also achieved. We suggest that this method will be a useful tool in elucidation of the chemical and biological properties of the proteins from chromatin.