INTERACTIONS OF AN ELECTRON-RICH TETRACATIONIC TENTACLE PORPHYRIN WITH CALF THYMUS DNA

The interactions between the water-soluble tentacle porphyrin meso-tet rakis[4-[(3-(trimethylammonio)propyl)- oxy]phenyl]porphine (T theta OP P) and calf thymus (CT) DNA had been found to exhibit unusual features : (a) At pH 7.0, T theta OPP underwent extensive self-stacking along t he DNA surface (Marzilli, et al. J. Am. Chern. Soc. 1992, 114, 7575); (b) the DNA-bound form of T theta OPP was extensively protonated even at pH 7.0 (Petho, et al. J. Chem. Soc., Chem. Commun. 1993, 1547). In the studies presented here, the formation of the T theta OPP-DNA adduc t was investigated under various conditions of pH, salt concentration, [porphyrin]/[DNA base pair] ratio (R), and absence/presence of buffer . Under all conditions studied, T theta OPP was an outside binder. Met hods employed included circular dichroism (CD), UV-visible absorbance, and fluorescence spectrophotometry. Time-dependence experiments were also conducted in order to detect any change in binding characteristic s over time. Evidence for outside-bound, self-stacked forms of T theta OPP included a large conservative induced visible CD feature, whereas the evidence for DNA-bound, protonated T theta OPP included a Soret b and red-shifted to 451 nm, a single positive induced CD band at 451 nm , and a red-shifted fluorescence band at 740 nm. At least two forms of the outside-bound, self-stacked porphyrin were evident. At high R (0. 25), hypochromicity of the Soret band and a conservative type CD featu re indicated that T theta OPP was extensively stacked. At lower R (0.0 5), increases in the Soret band intensity and changes in the band shap e suggested a less extensively stacked form, but this form of T theta OPP had a larger conservative feature in the CD spectrum. The intensit y of the conservative band suggested that the less stacked form is ver y stable at R = 0.05. The CD data are consistent with a model in which the interacting T theta OPP in the less stacked form has a different relative orientation than the more stacked form. Both high salt concen tration and PIPES buffer appeared to stabilize the outside-bound, self -stacked form of T theta OPP. At R = 0.01, a decrease in CD band inten sity suggested that the porphyrin was less stacked than at R = 0.05; t his change in stacking occurred over 1-2 h. Under these low R conditio ns where outside-bound self-stacking is less favored, the degree of T theta OPP protonation is highest. The conservative-type CD spectrum ch anged to a simple positive CD band upon protonation of T theta OPP, in dicating that the binding mode underwent a transition from outside bin ding with self-stacking to simple outside binding. High salt concentra tion (100 mM NaCl) was found to decrease the degree of protonation, an d the presence of PIPES buffer also decreased the degree of protonatio n at pH 7.0. Under conditions which favored protonation (low salt or n o PIPES), the degree of protonation was found to increase gradually du ring the first few hours after sample preparation. Since in the time c ourse of the experiment the total contents of the solution were unchan ged, and since the isolated unstacked porphyrin should protonate quick ly, these results suggest that stacking inhibits protonation of the DN A-bound porphyrin.