Human Spleen Histone H2B

Isolation and Amino Acid Sequence

Abstract

The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and HI, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments, I to IV, by limited chymotryptic digestion at pH 5.0 and 15°C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1-40, 41-42, 43-121, and 122-125 of the total sequence. The only new findings were microhetero-geneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30 alanine). The sequence of the most basic cluster at residues 27-34, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.