The histones of the pea plant, Pisum sativum L., and of calf thymus have been fractionated and further characterized in order to determine the extent of heterogeneity and the main chemical features of these basic nuclear proteins. Histones were fractionated by chromatography on Amberlite CG-50 and by preparative disc electrophoresis. The resulting highly purified histone fractions were further characterized by analytical disc electrophoresis, amino acid analysis, N-terminal and C-terminal analyses, and the preparation of tryptic peptide maps. Calf thymus histones Ia, Ib, IIb1, III and IV (f1, f1, f2a2, f3, and f2a1 in the nomenclature of Johns, Phillips and Butler) and pea bud histones Ib, IIb, III and IV were obtained aselectrophoretically pure components and each appears to be a single molecular species on the basis of N-terminal and C-terminal analysis and the number of tryptic peptides. The total number of major histones in calf thymus appears to be six, in pea bud, eight. The apparent heterogeneity of calf thymus histones demonstrated by disc electrophoresis is largely due to the formation of histone III complexes by disulfide bridges between histone III monomers. While calf thymus histone III contains two cysteines per molecule pea bud histone III contains but one and thus can form only dimers.
For each calf thymus histone there appears to be an homologous pea bud histone. It is proposed that the homologous pea and calf histones are related by evolution and perform identical biological functions. This hypothesis is based upon remarkable similarities in chromatographic and electrophoretic behavior, amino acid compositions, terminal amino acids, and in some cases even peptide maps of corresponding pea and calf histones. Peptide maps of the arginine-rich histone III contain 29 soluble peptides of which 26 are common to calf and pea; maps of histone IV contain 32 peptides of which 27 are common to calf and pea.
By chromatography and electrophoresis the histones of various pea tissues are qualitatively identical to those of pea bud. There are, however, quantitative differences and these have been accurately measured by a method of quantitative analytical disc electrophoresis. Young pea cotyledons contain only about a third as much lysine-rich histone as do mature cotyledons. Exploratory experiments on the synthesis of histone in pea cotyledons as a function of development and in relation to other macromolecular parameters are described in an appendix.
The dissociation of histones from pea bud nucleohistone by NaCl was studied, employing quantitative disc electrophoresis. Histone I (lysine-rich) is selectively dissociated by 0.5 M NaCl and the remaining histones are non-selectively dissociated primarily over the range 0.5 - 1.5 M NaCl. These data are compared with data for the dissociation of calf thymus histones from nucleohistone by NaCl and the general similarities are noted.
