Structure Studies on Chromatin and Nucleohistones. Thermal Denaturation Profiles Recorded in the Presence of Urea

Allen T. Ansevin, Lubomir S. Hnilica, Thomas C. Spelsberg, Susan L. Kehm

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

Structural information about chromatin and artificial nucleohistones was revealed by plots of the temperature derivative of hyperchromicity vs. the temperature (derivative denaturation profiles) from thermal denaturation experiments. Soluble preparations of rat thymus or liver chromatin displayed derivative denaturation profiles with a characteristic pattern of ascending steps when studied in a medium containing 3.6 m urea. Such profiles provide a convenient test for the intactness of chromatin since degraded preparations showed distinctly different patterns, characterized by more sharply defined maxima and minima. Comparisons of denaturation profiles of treated and untreated nucleoproteins in various denaturation media revealed that: (1) profiles of natural chromatin were more complex than those of artificial nucleohistones; (2) less than 10% of the nucleic acid within fresh chromatin denatured as extended stretches of free DNA; (3) both autolysis of chromatin and trypsin digestion exposed DNA and destabilized distinctive regions in the denaturation profile; (4) fixation of chromatin by formaldehyde elevated denaturation temperatures; (5) derivative denaturation profiles appear to reflect physical as well as chemical features of nucleoprotein complexes; (6) a cacodylate buffer containing 3.6 M urea resolves the structure of chromatin better than a buffer containing only 5 HIM sodium cacodylate, and reveals the structure of lysine-rich nucleohistones better than 0.25 mM Versenate buffer; (7) spontaneous formation of ammonium cyanate in a urea-containing medium does not produce major artifacts that prevent the interpretation of denaturation profiles; (8) the nucleohistones reconstituted at low ionic strength in this laboratory appeared to have greater structural variety than related complexes reconstituted elsewhere by the gradient dialysis method.

Original languageEnglish (US)
Pages (from-to)4793-4803
Number of pages11
JournalBiochemistry
Volume10
Issue number25
DOIs
StatePublished - Dec 1 1971

ASJC Scopus subject areas

  • Biochemistry

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