Accessibility of nucleic acid-complexed biomolecules to hydroxyl radicals correlates with their conformation: A fluorescence polarization spectroscopy study

A fluorescence methodology has been developed to examine the relationship between the conformational state of specific biomolecules in simple chromatin models and their accessibility to hydroxyl radicals (^.OH). Polylysine and histone H1 were labelled with SECCA, the succinimidyl ester of coumarin-3-carboxylic acid, which generates the fluorescent derivative 7-OH-SECCA following its interaction with radiation-induced ^.OH in aqueous solution. The fluorescence induced per unit γray dose reflecting the accessibility of ^.OH to such SECCA-conjugated biomolecules was recorded. The biomolecules were also labelled with the fluorescent derivative 7-OH-SECCA in trace amounts to study their conformation under identical conditions via fluorescence polarization spectroscopy. When these biomolecules were complexed with a polynucleotide or DNA, a major increase in polarization anisotropy was recorded. Upon salt-induced dissociation of these biomolecules from the nucleic acids, the increase in anisotropy was reversed. The histone H1-DNA complexes also exhibited an initial increase in anisotropy with increasing NaCl concentration (maximum at 100 mmol dm^-3) indicating the possible formation of a more compact conformation. The fluctuations in anisotropy were inversely proportional to the recorded fluorescence/Gy. The data indicate a direct correlation between the accessibility of ^.OH to polylysine or histone H1 complexed with nucleic acids and the conformation of these biomolecules.