Poulos A T, Kuzmin V, Geacintov N E
J Biochem Biophys Methods. 1982 Sep;6(4):269-81. doi: 10.1016/0165-022x(82)90009-4.
Triplet flash photolysis techniques, coupled with quenching of the triplets by molecular oxygen, are utilized as probes of the microenvironment of polycyclic aromatic molecules bound covalently and non-covalently to DNA. The triplet-oxygen quenching properties of the following adducts in aqueous solutions at 25 +/- 1 degrees C were investigated: covalent adducts derived from the reaction of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BaPDE) and of (+/-)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE) with DNA, and non-covalent intercalation complexes of acridine orange (AO) and DNA. In all cases the quenching follows the Stern-Volmer quenching law with a quenching constant of KTO2 approximately equal to 10(9) M-1 X S-1 for the covalent BaPDE-DNA and BaPE-DNA complexes in aqueous solution. This value of KTO2 is characteristic of free molecules (not bound to DNA) and indicates that the pyrene chromophore is totally accessible to oxygen, and is thus not located at an intercalation-type of binding site in these covalent adducts. In contrast, the AO-DNA complexes are characterized by values of KTO2 approximately equal to 10(8) M-1 X S-1 indicating that the intercalated AO molecules are about ten times less accessible to molecular oxygen than free AO molecules. The KTO2 values for the covalent BaPDE-DNA and BaPE-DNA adducts decrease when the DNA concentration is increased in the 1 X 10(-4)-3 X 10(-3) M range (expressed in nucleotide concentration). This effect is attributed to intermolecular DNA-DNA interactions in which segments of adjacent DNA molecules tend to cover the pyrene chromophores on other strands, thus decreasing their accessibility to oxygen. In contrast the values of KTO2 for the non-covalent AO-DNA intercalation complexes are independent of DNA concentration, as expected for interior binding sites.
三线态闪光光解技术，结合分子氧对三线态的猝灭作用，被用作探测与DNA共价结合和非共价结合的多环芳烃分子微环境的探针。研究了在25±1℃的水溶液中下列加合物的三线态氧猝灭性质：由（±）-7β,8α-二羟基-9α,10α-环氧-7,8,9,10-四氢苯并[a]芘（BaPDE）和（±）-9,10-环氧-7,8,9,10-四氢苯并[a]芘（BaPE）与DNA反应生成的共价加合物，以及吖啶橙（AO）与DNA的非共价插入复合物。在所有情况下，猝灭都遵循Stern-Volmer猝灭定律，对于水溶液中的共价BaPDE-DNA和BaPE-DNA复合物，猝灭常数KTO2约等于10(9) M-1×s-1。KTO2的这个值是游离分子（未与DNA结合）的特征，表明芘发色团完全可被氧接触，因此在这些共价加合物中它不位于插入型结合位点。相反，AO-DNA复合物的特征是KTO2值约等于10(8) M-1×s-1，这表明插入的AO分子比游离AO分子对分子氧的可及性大约低十倍。当DNA浓度在1×10(-4)-3×10(-3) M范围内（以核苷酸浓度表示）增加时，共价BaPDE-DNA和BaPE-DNA加合物的KTO2值降低。这种效应归因于分子间DNA-DNA相互作用，其中相邻DNA分子的片段倾向于覆盖其他链上的芘发色团，从而降低它们对氧的可及性。相反，非共价AO-DNA插入复合物的KTO2值与DNA浓度无关，这对于内部结合位点来说是预期的。
