FLUORESCENCE STUDY OF THE PHYSICO‐CHEMICAL PROPERTIES OF A BENZO(A)PYRENE7,8‐DIHYDRODIOL9,10‐OXIDE DERIVATIVE BOUND COVALENTLY TO DNA

Abstract— Covalent complexes between7,8‐dihydrodiol 9.10‐oxide benzo(a)pyrene (BPDE) and DNA with a modification of one BPDE molecule per 1000 DNA bases were prepared in vitro. The same stereoselective and chemically homogeneous binding of BPDE to native DNA was observed, as reported earlier for human and bovine bronchial explants. The fluorescence of the pyrene‐like aromatic moiety of BPDE bound to DNA in vitro was used as a probe of the microenvironment of the BPDE molecule in order to obtain information about the structure of the BPDE‐DNA complex dissolved in aqueous solution. Fluorescence techniques, based on the quenching of the singlet excited states by metal ions such as Ag⁺, by iodide ions, and by molecular oxygen are described, which provide a method for differentiating between external and internal (intercalation) binding of polycyclic aromatic molecules to DNA. Silver ions, which bind specifically to DNA bases, exhibit a strong quenching effect on noncovalently bound, intercalated benzo(a)pyrene; on the other hand, there is no quenching effect on the fluorescence of BPDE in the covalent DNA adduct. Quenchers such as O₂ and iodide ions, which do not specifically bind to DNA and are dissolved in the solution external to the DNA molecule, exhibit a quenching effect on the BPDE chromophore. Furthermore, the fluorescence yield of the BPDE‐DNA complex decreases with increasing DNA concentration, an effect which is not observed with non‐covalently bound intercalated benzo(a)pyrene‐DNA complexes, and which is attributed to intermolecular DNA‐DNA interactions. The results of these studies indicate that the pyrene‐like chromophore in the covalent BPDE‐DNA complex is not intercalated between the base pairs, and that it is located in an accessible region external to the DNA helix. Possible structures are discussed.