Fractionation of Calf Thymus Histone by a New Method of CM-cellulose Chromatography*

A new chromatographic method has been developed for the fractionation of calf thymus histone. The method consists of the elution of histone from a CM-cellulose column with a linearly decreasing gradient of ethanol in 2.6 M formic acid containing 0.1 M sodium formate. A unique elution order was found in that the fractions were eluted in the order of increasing lysine/arginine ratio. With the whole histone, resolution of the earlier eluted arginine-rich histones is insufficient, but the later eluted slightly lysine-rich and very lysine-rich histones are clearly separated. The fractionation can be made more efficient by using as starting materials the histone fractions F1, F2a, F2b, and F3 obtained by the method of Johns. F2a is resolved into three subfractions, two of which are distinguishable by rechromatography but almost identical in their glycine-rich, arginine-rich composition and single-band disc-electrophoretic pattern. The third subfraction is an intermediate type having equimolar contents of arginine and lysine. A typical slightly lysine-rich histone is obtained from F2b as a main fraction. It behaves as a single component in rechromatography, disc electrophoresis, and terminal amino acid analyses. Another subfraction similar to the intermediate-type subfraction of F2a is also found. The possibility of subfractionation of F1 is also observed. The present method is least effective in further fractionation of F3.