Studies of the binding of diolepoxide metabolites of polycyclic aromatic hydrocarbons to DNA using electrofluorescence polarization spectroscopy.

In the electrofluorescence method, a solution of DNA with covalently bound polycyclic hydrocarbons is placed in an electric field, and changes in the intensity of polarized fluorescence are observed. Under the correct conditions, these charges can be used to determine a value for the angle psi between the long axis of the hydrocarbon molecule and the axis of the DNA helix. For DNA or poly(dA-dT) treated with each stereoisomer of anti-benzo[c]phenanthrene diolepoxide, psi ranged from 55 degrees to 61 degrees, consistent with a mixture of quasi-intercalated adenine adducts and externally bound guanine adducts. Similar results were obtained with another set of 'fjord-region' diolepoxides, derived from benzo[c]chrysene. Adducts in DNA treated with diolepoxides derived from chrysene, 5-methylchrysene or 6-methylchrysene gave psi of about 53 degrees, so the predominant adducts are externally bound, probably in the minor groove of DNA.

Binding of biologically important molecules to DNA, probed using electro-fluorescence polarization spectroscopy.

When small fluorescent tag molecules are bound with a single geometry to larger carrier molecules the fluorescence excited using polarized light may itself be partially polarized. If this is the case then alignment of the carrier molecules changes the fluorescence polarization. Electro-fluorescence polarization spectrometry (EFPS) makes use of the fluorescence polarization changes which occur when the carrier molecules are aligned by an electric field. EFPS allows the determination of the binding geometry of the small tag molecules to the larger carrier molecules. In this study, EFPS has been used to probe the interactions between DNA and a series of chromosomal stains and anti-neoplastic (anti-cancer) agents. Results are presented for calf thymus DNA, treated with the dyes acridine orange, ethidium bromide, proflavine, Hoechst 33258, Hoechst 33342, 4',6-diamidino-2-phenylindole and the anti-tumour agents, doxorubicin, daunomycin, actinomycin C and actinomycin V.