Disulfide bonds and the structure of the chromatin complex in relation to aging

Triton X-100 washed nuclei from livers of young-adult and old mice were digested with micrococcal nuclease and pelleted. Supernatants (1SF) were saved and the pellets lysed in a hypotonic EDTA buffer. A second supernatant (2SF) and a final pellet (P) were obtained by recentrifugation (7000 g, 7 minutes). The 1SF/2SF ratio, which has been shown to be an index of the transcriptionally active to inactive chromatin ratio, was lower in older mice. The fraction relatively resistant to solubilization by the nuclease (P) was found by isopycnic sucrose gradient centrifugation to be in a more compatc, condensed state when prepared from older mice. Higher amounts of heavy density chromatin were obtained from nuclei of old than young mice by hypotonic lysis plus minimal mechanical shearing. 2-Mercaptoethanol (2ME) treatment brought the density of the material of P from old mice back to the levels of young mice. In both age groups 2ME decreased the densities of mechanically sheared chromatin as well as of the whole Triton X-100 washed nuclei. In nuclease digestion experiments treatment of the nuclei from both age groups with S-S reducing agents increased the release of DNA from P into the supernatants. The results are consistent with S-S bonds being involved in the condensed structure of chromatin in young and old mice and in the shift of the chromatin complex towards a more compact, condensed state in old age.