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 relations hip between the conformational state of specific biomolecules in simpl e 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 d erivative 7-OH-SECCA following its interaction with radiation-induced .OH in aqueous solution. The fluorescence induced per unit gamma-ray d ose reflecting the accessibility of .OH Co such SECCA-conjugated biomo lecules was recorded. The biomolecules were also labelled with the flu orescent derivative 7-OH-SECCA in trace amounts to study their conform ation under identical conditions via fluorescence polarization spectro scopy. When these biomolecules were complexed with a polynucleotide or DNA, a major increase in polarization anisotropy was recorded. Upon s alt-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 fo rmation 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 pol ylysine or histone H1 complexed with nucleic acids and the conformatio n of these biomolecules.