NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer.
ABSTRACT 500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.
Article: Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.[show abstract] [hide abstract]
ABSTRACT: Hydroethidine (HE; or dihydroethidium) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E(+)). In biological systems, another red fluorescent product, ethidium, is also formed, usually at a much higher concentration than 2-OH-E(+). In this article, we review the methods to selectively detect the superoxide-specific product (2-OH-E(+)) and the factors affecting its levels in cellular and biological systems. The most important conclusion of this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E(+), using confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E(+), to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted hydroethidine (Mito-HE, MitoSOX red) with superoxide is similar to the reactivity of HE with superoxide, and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or MitoSOX) as well.Free radical biology & medicine 04/2010; 48(8):983-1001. · 5.42 Impact Factor