Identification of nonhistone chromatin proteins in chromatin subunits

Rat liver chromatin was digested by micrococcal nuclease. More than 80% of the enzyme-digested chromatin could be recovered after centrifugation. Treatment with sodium deoxycholate and Triton X-100 at concentrations of 0.5% in the final chromatin suspension gave a higher recovery. Chromatin subunits were fractionated on a 5-30% linear sucrose density gradient. Approximately 35% of the chromatin subunits could be recovered from the gradient. Chromatin subunits and their DNA fragments were identified by gel electrophoresis and ultracentrifugation. The presence of nonhistone chromatin proteins (NHCP) in chromatin subunits was demonstrated by the following criteria: (i) Quantitative analysis showed that the mass ratio of histone to NHCP, in the presence or absence of detergents, was 1:0,25 or 1:0.1, respectively. (ii) After the removal of acid-soluble protein from the subunits, it was found that most of the phenol-soluble NHCP were similar to total chromatin NHCP. However, four major fractions of these phenol-soluble NHCP were found to be enriched in the subunits as identified by two-dimensional polyacrylamide gel electrophoresis. (iii) Experiments using an exchange of isotope-labeled and nonlabeled chromatin showed that NHCP were tightly bound to the chromatin subunits.