|Abstract or Summary
- Histones are presumed to be of chromosomal origin and provide
both structural and functional elements for chromatin. In like
manner the ribosomal proteins are of ribosomal origin and have
analogous roles. Considerable controversy over their cellular roles
exists because of the inability to compare directly both groups of
proteins. In order to compare definitively the histone with the ribosomal
proteins: 1) the organelles from which they are derived must
be prepared under conditions which eliminate cross contamination
and 2) a preparative scale method must be developed to fractionate
both histone and ribosomal protein under similar conditions.
A chromatographic method was developed which fractionated
histone and ribosomal protein simultaneously. The histone and
ribosomal proteins were isolated from beef and rat liver and germinating pea roots. Histone was acid extracted from exhaustively washed chromatin prepared from purified nuclei. Ribosomal protein was
extracted from purified ribosomes. Both groups of protein were
adsorbed to a column of carboxymethyl cellulose: and eluted with a
sodium acetate gradient (0.01 M to 0.4 M, pH 5.6) in 6 M urea.
This method is superior to IRC-50 chromatography for the following
reasons: 1) no irreversible adsorption, 2) both histones and ribosomal
proteins elute under the same conditions, 3) all fractions elute with a
single uninterrupted gradient, 4) the reagents are more conveniently
prepared and 5) the arginine-rich histones can be resolved into fractions
III and IV,
Beef liver and pea root histones were fractionated into six peaks
corresponding in appearance to the fractions Ia, Ib, Ha, IIb, IIc and
III - IV of conventional IRC-50 chromatography. Rat liver histone
lacked both Ib and IIc, but peak III was resolved from peak IV with
a 400 ml gradient. Ribosomal proteins were fractionated into 13
peaks (rat liver), 22 peaks (beef liver) and 17 peaks (pea root) in
agreement with the number obtained from polyacrylamide gel elctrophoresis.
When assayed turbidimetrically some of the histones
and ribosomal proteins exhibited chromatographic identity, A
spectrofluorometric assay indicated differences between chromatographically
identical histone and ribosomal protein fractions.
Histones and ribosomal proteins, while possessing similar
physical and chemical properties, appear to have unique cellular
origins and distinct roles.