Shirley C.M. Gandini

Photophysical studies of excited-state characteristics of meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin bound to DNA

The interaction of meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin (TMPyP) with DNA has been investigated at ionic strength (IS) values of 0.01 and 0.20 M (pH 6.8) using the flash-photolysis technique along with optical absorption and fluorescence data. It is found that the aggregation of TMPyP observed at the porphyrin excess reduces its total fluorescence intensity (JT) and the T-T absorption. At low IS the JT and the T-T absorption for the TMPyP monomers bound to the GC DNA sites are lower than those for the free ones, whereas binding to the AT sites (high IS) increases JT. At low IS the triplet decay of TMPyP is mono-exponential, the lifetime increasing with the [DNA] increase, while at high IS the addition of DNA transforms this profile to a bi-exponential form with lifetimes of the components independent of [DNA]. Binding to DNA reduces the quenching constants of the porphyrin triplet states by molecular oxygen (kq), the effect depending on the site and mode of binding. So, at low IS the kq value for the TMPyP externally bound to the GC sites (3.0 x 10(8) M-1 s-1) is five times lower than that for the free porphyrin and twice as high as that for the intercalated one. At high IS the TMPyP binding reduces the kq three-fold for the AT sites in the minor groove and 16-fold in the major groove as compared with the free one (approximately or equal to 1.6 x 10(9) M-1 s-1).

Interaction of Fe(III)- and Zn(II)-tetra(4-sulfonatophenyl) porphyrins with ionic and nonionic surfactants: aggregation and binding.

Interactions of the water soluble Fe(III)- and Zn(II)-tetra(4-sulfonatophenyl) porphyrins, FeTPPS(4) and ZnTPPS(4), with ionic and nonionic micelles in aqueous solutions have been studied by optical absorption, fluorescence, resonance light-scattering (RLS), and 1H NMR spectroscopies. The presence of three different species of both Fe(III)- and Zn(II)TPPS(4) in cationic cetyltrimethylammonium chloride (CTAC) solution has been unequivocally demonstrated: free metalloporphyrin monomers or dimers (pH 9), metalloporphyrin monomers or aggregates (possibly micro-oxo dimers) bound to the micelles, and nonmicellar metalloporphyrin/surfactant aggregates. The surfactant:metalloporphyrin ratio for the maximum nonmicellar aggregate formation is around 5-8 for Fe(III)TPPS(4) both at pH 4.0 and 9.0; for Zn(II)TPPS(4) this ratio is 8, and the spectral changes are practically independent of pH. In the case of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and non-ionic polyoxyethylene lauryl ether (Brij-35) and t-octylphenoxypolyethoxyetanol (Triton X-100), the nonmicellar aggregates were not observed in the pH range from 2.0 to 12.0. Binding constants were calculated from optical absorption data and are of the order of 10(4) M(-1) for both CTAC and HPS, values which are similar to those previously obtained for the porphyrin in the free base form. For Brij-35 and Triton X-100 the binding constant for ZnTPPS(4) at pH 4.0 is a factor of 3-5 lower than those for CTAC and HPS, while in the case of FeTPPS(4) they are two orders of magnitude lower. Our data show that solubilization of ZnTPPS(4) within nonpolar regions of micelles is determined, in general, by nonspecific hydrophobic interactions, yet it is modulated by electrostatic factors. In the case of FeTPPS(4), the electrostatic factor seems to be more relevant. NMR data indicated that Fe(III)TPPS(4) is bound to the micelles predominantly as a monomer at pH 4.0, and at pH 9.0 the bound aggregated form (possibly micro-oxo dimers) remains. The metalloporphyrins were incorporated into the micelles near the terminal part of their hydrocarbon chains, as evidenced by a strong upfield shift of the corresponding peaks of the surfactants.

Aggregation of meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin in its free base, Fe(III) and Mn(III) forms due to the interaction with DNA in aqueous solutions: Optical absorption, fluorescence and light scattering studies

Interactions of the water soluble meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin (TMPyP) in its free base, Mn(III) and Fe(III) forms with DNA in aqueous solutions have been studied by optical absorption, fluorescence and resonance light-scattering (RLS) spectroscopies. Optical absorption and fluorescence measurements have demonstrated the presence of three different species of TMPyP free base and its Mn(III) form in DNA solutions. These species correspond to free porphyrin monomers, monomers bound to DNA and porphyrin aggregates formed on the surface of DNA molecules. This assignment correlates very well with the RLS data. Aggregation reduces the fluorescence of the TMPyP free base. Fe(III)TMPyP also forms aggregates, however, more than three species exist in the presence of DNA due to the equilibria between its free and bound monomers and μ-oxo dimers. The degree of aggregation of Mn(III) and Fe(III) forms of TMPyP is higher than that of its free base. One of the possible explanations of this fact lies in the competition between intercalation and aggregation of TMPyP free base in the process of its binding to DNA; the intercalation of porphyrin should reduce its degree of aggregation. For the Mn(III) and Fe(III) TMPyP forms this competition does not exist as they do not intercalate.

Binding of the Mn(III) complex of meso-tetrakis(4-N-methyl-pyridimumyl) porphyrin to DNA. Effect of ionic strength

Interactions of the water-soluble Mn(III) complex of meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin (Mn(III)TMPyP) with DNA in aqueous solutions at low (0.01 M) and high (0.2 M) ionic strengths have been studied by optical absorption, resonance light scattering (RLS) and 1H NMR spectroscopies. Optical absorption and RLS measurements have demonstrated that in DNA solutions at low ionic strength the Mn(III)TMPyP form aggregates, which are decomposed at DNA excess. At high ionic strength the aggregation was not observed. We explain this effect by assuming that upon increase in ionic strength, Mn(III) TMPyP dislocates from the DNA sites, which produces better conditions for the porphyrin aggregation, to sites where the aggregation is hindered. The 1H NMR data demonstrated that the aggregation observed at low ionic strength reduces the paramagnetism of Mn(III)TMPyP. This phenomenon was not observed at the high ionic strength in the absence of aggregation.

Spectroscopic study of a water-soluble iron(III) meso-tetrakis(4-N-methylpyridiniumyl) porphyrin in aqueous solution: effects of pH and salt.

The equilibrium behavior of cationic iron(III) meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin, Fe(III)TMPyP, in aqueous solution was studied as a function of pH by optical absorption, EPR and (1)H NMR spectroscopies. The presence of several Fe(III)TMPyP species in solution was unequivocally demonstrated: monomeric porphyrin species (a monoaqueous five-coordinated complex, a diaaqueous six-coordinated complex and a monoaqueous-hydroxo six-coordinated complex), a micro-oxo dimer and a bis-hydroxo complex. The addition of salt to the porphyrin solution leads to a simplification of the equilibrium as a function of pH. In this case, only three species were observed in solution: a monomeric porphyrin species, a micro-oxo dimer and a bis-hydroxo complex. Optical absorption, EPR and (1)H NMR spectra contributed to the characterization of these species. Four critical pH values (pK) for Fe(III)TMPyP were obtained in pure buffer and only three pK values were observed in the presence of NaCl. The addition of salt favors the presence of the dimeric species in solution and simplifies the equilibrium in the acidic pH range.

On the localization of water-soluble porphyrins in micellar systems evaluated by static and time-resolved frequency-domain fluorescence techniques

Fluorescence quenching of meso-tetrakis-4-sulfonatophenyl (TPPS(4)) and meso-tetrakis-4-N-methylpyridil (TMPyP) porphyrins is studied in aqueous solution and upon addition of micelles of sodium dodecylsulfate (SDS), cetyltrimethylammonium chloride (CTAC), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and t-octylphenoxypolyethoxyethanol (Triton X-100). Potassium iodide (KI) was used as quencher. Steady-state Stern-Volmer plots were best fitted by a quadratic equation, including dynamic (K(D)) and static (K(S)) quenching. K(S) was significantly smaller than K(D). Frequency-domain fluorescence lifetimes allowed estimating bimolecular quenching constants, k(q). At 25 degrees C, in aqueous solution, TMPyP shows k(q) values a factor of 2-3 higher than the diffusional limit. TPPS(4) shows collisional quenching with pH dependent k(q) values. For TMPyP quenching results are consistent with reported binding constants: a significant reduction of quenching takes place for SDS, a moderate reduction is observed for HPS and almost no change is seen for Triton X-100. Similar data were obtained at 50 degrees C. For CTAC-TPPS(4) system an enhancement of quenching was observed as compared to pure buffer. This is probably associated to accumulation of iodide at the cationic micellar interface. The attraction between CTAC headgroups and I(-), and repulsion between SDS and I(-), enhances and reduces the fluorescence quenching, respectively, of porphyrins located at the micellar interface. The small quenching of TPPS(4) in Triton X-100 is consistent with strong binding as reported in the literature.

Spectroscopic studies of the interaction of cationic water-soluble iron(III) meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (FeTMPyP) with ionic and nonionic micelles

Interactions of cationic FeTMPyP with ionic and nonionic micelles have been studied by optical absorption, resonance light scattering (RLS) and 1H NMR spectroscopies. The equilibrium behavior of FeTMPyP as a function of pH is described by several species in aqueous solution. The presence of phosphate anions leads to the existence of additional species in the acid pH region, probably due to the coordination of phosphates to the iron. FeTMPyP solution as a function of pH in the presence of anionic SDS showed a simplified equilibrium in acidic pH region, favoring the transition to the dimeric species. Titration of FeTMPyP as a function of SDS surfactant concentration showed the presence of three different porphyrin species: free metalloporphyrin monomers (or dimers depending on pH), metalloporphyrin monomers (or dimers) bound to the micelles, and nonmicellar metalloporphyrin/surfactant aggregates. In the case of zwitterionic LPC and HPS, and nonionic TRITON X-100 the nonmicellar metalloporphyrin/surfactant aggregates were not observed. Binding constants were calculated from optical absorption data and have values of 2 × 103M−1 for SDS being much smaller for HPS (58 M−1), LPC and TRITON X-100. Comparison with our previous data for anionic FeTPPS4 shows that both the electrostatic factor and hydrophobic forces are relevant in the porphyrin-surfactant interaction: for FeTPPS4 binding constants to cationic CTAC and zwitterionic HPS are of the same order of magnitude, 1-3 × 104M−1; for FeTMPyP the delocalization of the positive charges from the periphery substituents into the macrocycle ring leads to reduction of both electrostatic attraction to the micelle as well as hydrophobic character of the porphyrin ring, leading to a 10-fold reduction of binding to the micelles of opposite charge to the porphyrin. NMR data indicated that FeTMPyP is bound to the micelles as an equilibrium of two forms of monomer at pH 2.0, and at pH 9.0 the bound aggregated form (possibly dimers) is observed predominantly with some amount of a monomeric form.