Interactions of an Electron-Rich Tetracationic Tentacle Porphyrin with Calf Thymus DNA

Abstract

The interactions between the water-soluble tentacle porphyrin meso-tetrakis[4-[(3-(trimethylammonio)propyl)oxy]phenyl]porphine (TBOPP) and calf thymus (CT) DNA had been found to exhibit unusual features: (a) At pH 7.0, TBOPP underwent extensive self-stacking along the DNA surface (Marzilli, et al. J . Am. Chem. SOC. 1992, 114, 7575); (b) the DNA-bound form of TBOPP 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 TBOPP-DNA adduct was investigated under various conditions of pH, salt concentration, [porphyrin]/[DNA base pair] ratio (R) , and absence/presence of buffer. Under all conditions studied, TBOPP was an outside binder. Methods employed included circular dichroism (CD), UV-visible absorbance, and fluorescence spectrophotometry. Timedependence experiments were also conducted in order to detect any change in binding characteristics over time. Evidence for outside-bound, self-stacked forms of TBOPP included a large conservative induced visible CD feature, whereas the evidence for DNA-bound, protonated TBOPP included a Soret band red-shifted to 451 nm, a single positive induced CD band at 45 1 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 feature indicated that TBOPP was extensively stacked. At lower R (0.05), increases in the Soret band intensity and changes in the band shape suggested a less extensively stacked form, but this form of TBOPP had a larger conservative feature in the CD spectrum. The intensity of the conservative band suggested that the less stacked form is very stable at R = 0.05. The CD data are consistent with a model in which the interacting TBOPP in the less stacked form has a different relative orientation than the more stacked form. Both high salt concentration and PIPES buffer appeared to stabilize the outside-bound, self-stacked form of TBOPP. At R = 0.01, a decrease in CD band intensity suggested that the porphyrin was less stacked than at R = 0.05; this change in stacking occurred over 1-2 h. Under these low R conditions where outside-bound self-stacking is less favored, the degree of TBOPP protonation is highest. The conservative-type CD spectrum changed to a simple positive CD band upon protonation of TBOPP, indicating that the binding mode underwent a transition from outside binding with self-stacking to simple outside binding. High salt concentration (100 mh4 NaCl) was found to decrease the degree of protonation, and the presence of PIPES buffer also decreased the degree of protonation at pH 7.0. Under conditions which favored protonation (low salt or no PIPES), the degree of protonation was found to increase gradually during the first few hours after sample preparation. Since in the time course of the experiment the total contents of the solution were unchanged, and since the isolated unstacked porphyrin should protonate quickly, these results suggest that stacking inhibits protonation of the DNA-bound porphyrin.