Iouri E. Borissevitch

Fluorescence and optical absorption study of interaction of two water soluble porphyrins with bovine serum albumin. The role of albumin and porphyrin aggregation

Abstract The interaction of two metal-free water soluble porphyrins (PPh), meso-tetrakis (p-sulfo-natophenyl)porphyrin (TPPS4) and meso-tetrakis(4-N-methyl-pyridiniumyl)porphyrin (TMPyP), with bovine serum albumin (BSA) was investigated in the pH range from 4.0 to 8.5 using optical absorption and fluorescence spectroscopies. It was found that in this pH range both porphyrins bound to BSA exist in deprotonated free base forms. The binding of PPh quenches the BSA fluorescence. On the contrary, the fluorescence of both monomeric porphyrins increases by the binding. Two types of aggregates were found: those of BSA, and the TPPS4 aggregates on the surface of the BSA molecule. The TPPS4 aggregation was observed only when its concentration was higher than that of BSA ( [ TPPS 4 ] [ BSA ] > 1 ), the fluorescence of TPPS4 being reduced by its aggregation. The TMPyP does not form aggregates. A step-by-step aggregation model was developed to determine the average aggregation numbers of both the BSA (〈j〉) and the TPPS4 ( k 〉 ) from the fluorescence quenching. The (〈j〉) values vary with pH, BSA concentration and the type of porphyrin from 3 ± 1 to 15 ± 3. The ( k 〉 ) value is 10 ± 2 at pHs 4.0 and 5.0 and 3 ± 1 at pH 8.5. Binding constants of PPh to BSA (Kh) are determined as the Stern-Volmer quenching constants of BSA fluorescence. However, the aggregation distorts the binding constant and its real value can be obtained as a limit of the Stern-Volmer one at the lowest possible BSA and PPh concentrations. The Kb values depend both on the charge and structure of porphyrin molecules and on the charge and/or the conformation of BSA. The Kb values are for TPPS4 1.5 × 108 M−1 at pHs 4.0 and 5.0 and 3.2 × 106 M− at pH 8.5 and for TMPyP 7.3 × 105 M−1 at pH 5.0 and 1.8 × 106 M−1 at pH 8.5.

Photophysical studies on the interaction of two water-soluble porphyrins with bovine serum albumin. Effects upon the porphyrin triplet state characteristics

Abstract The interaction of two metal-free water soluble porphyrins (PPh): meso -tetrakis ( p -sulfo-natophenyl)porphyrin (TPPS 4 ) and meso -tetrakis(4- N -methyl-pyridiniumyl)porphyrin (TMPyP), with bovine serum albumin (BSA) was investigated in the pH range from 4.0 to 8.5 using the flash-photolysis technique in comparison with results obtained by optical absorption, fluorescence and resonance light scattering. It was found that in the presence of BSA, TPPS 4 can exist in aqueous solutions as free monomers, aggregates and/or monomers bound to a single BSA molecule and monomers inside the BSA aggregates, while TMPyP does not form aggregates at binding. Binding to BSA transforms the profile of the triplet decay curve from monoexponential to biexponential form with the component lifetimes and relative amplitudes depending on binding characteristics. The triplet lifetime of a bound porphyrin monomer is longer than that of a free one. The aggregation of TPPS 4 observed at [TPPS 4 ]/[BSA] > I reduces the T-T absorption since the lifetimes of the aggregate excited states are very short and/or quantum yield of the aggregate triplet state is very low. The porphyrin binding to BSA reduces the quenching constants of the porphyrin triplet states by molecular oxygen due to obstacles produced by binding. This effect is especially pronounced for the porphyrin molecules located inside BSA aggregates formed around the porphyrin molecules in excess BSA.

Resonance light scattering study of aggregation of two water soluble porphyrins due to their interaction with bovine serum albumin

Abstract The interaction of the water soluble meso -tetrakis( p -sulfonato-phenyl)porphyrin (TPPS 4 ) and meso -tetrakis(4- N -methyl-pyridiniumyl)porphyrin (TMPyP) with bovine serum albumin (BSA) in aqueous solutions has been studied by optical absorption, fluorescence and resonance light scattering spectroscopies. The formation of two types of aggregates due to this interaction has been demonstrated: aggregates of the TPPS 4 on the BSA molecule surface and aggregates of BSA molecules around the TPPS 4 molecule. The reduction of integral fluorescence intensity of TPPS 4 due to the porphyrin aggregation and its increase due to the BSA aggregation have been demonstrated. The influence of the porphyrin charge on the aggregation process has been also clearly observed and explained on the basis of known BSA binding properties.

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).

Binding of manganese and iron tetraphenylporphine sulfonates to albumin is relevant to their contrast properties

The interaction of Fe(III) and Mn(III) complexes of TPPS4 with bovine serum albumin (BSA) was studied by T1 relaxation measurements of water protons and high resolution 1H NMR of the porphyrin moieties. At excess of BSA, both metalloporphyrins bind to BSA as the high spin monomers. The relaxivity of bound MnTPPS4 is significantly higher as compared to the free form in solution. When metalloporphyrins are in excess, they aggregate at the BSA surface, up to two MnTPPS4, and up to 10-15 FeTPPS4 units per BSA globule. Bound aggregates are unable to enhance magnetic relaxation of water protons due to the antiferromagnetic coupling between metal ions in the aggregates. Therefore, the dose-effect dependences for metalloporphyrins in the range of metalloporphyrin/BSA ratio of 0 to 25 at the constant BSA concentration at pH 7.4 are characterized by a local maximum at about 2 for MnTPPS4, and a global maximum at about 3 for FeTPPS4, MnTPPS4 complex is more effective than FeTPPS4 in the whole concentration range. It is suggested that the difference in binding and aggregation properties of metalloporphyrins may be relevant to their relaxation efficiency in vivo, blood transport, and biodistribution.

AGGREGATION PHENOMENA IN THE COMPLEXES OF IRON TETRAPHENYLPORPHINE SULFONATE WITH BOVINE SERUM ALBUMIN

Binding of Fe(III) meso-tetrakis(p-sulfonatophenyl)-porphyrin (FeTPPS4) to bovine serum albumin (BSA) was studied by UV-VIS absorption, fluorescence quenching, circular dichroism, 1H NMR, and ESR. At excess of BSA, the bound form of FeTPPS4 is a high-spin monomer exhibiting a Soret band at 417 nm, a broad NMR peak at 10.3 ppm, an ESR signal at g = 5.7-5.9, and a strong enhancement of magnetic relaxation of water protons. In the intermediate concentration range, a formation of nonparamagnetic bound aggregates of FeTPPS4 occurs (up to 10-15 molecules at pH 6.0) with a Soret band at 414 nm and NMR peaks at 7.0, 8.1, and 12.7 ppm. In the physiologic pH range, BSA binds the monomeric form of FeTPPS4 with an association constant of about 10(8) M-1, the affinity to oxo-dimers in solution being much lower. BSA itself is also subject to aggregation with an average aggregation number of 4-8 in the physiological pH range. It is assumed that aggregation phenomena may play an important role, both in the relaxation efficiency of metalloporphyrins as MRI contrast agents and in the blood transport of porphyrin drugs by albumins.

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.

Spectroscopic studies of the interaction of bichromophoric cyanine dyes with DNA. Effect of ionic strength

Spectroscopic characteristics of a cyanine dye with two chromophores (biscyanine dye, BCD) in aqueous solutions and effects of NaCl and DNA upon these characteristics have been studied by optical absorption, circular dichroism (CD) and fluorescence spectroscopies. In homogeneous solutions, BCD is characterized by intense optical absorption (epsilon =1.33 x 10(5) M(-1) x cm(-1)) and weak fluorescence (phi(fl)=0.018) in the wavelength region greater than 600 nm. The dye forms H-aggregates at low concentrations (10(-6) M). NaCl stimulates the formation of both H- and J-aggregates of the dye at much lower dye concentrations, while DNA in low concentrations (<10(-6) M) stimulated the formation of just J-aggregates on the surface of the DNA molecule. Higher DNA concentrations induce the dye to disaggregate, and there exists an equilibrium between three dye forms: free monomers, J-aggregates and bound monomers, the maximum content of J-aggregates was observed at [DNA]/[BCD]=0.6+/-0.2 and total disaggregation at [DNA]/[BCD]=190+/-20. J-aggregates are characterized by phi(fl)=0.05 and bound monomers by phi(fl)=0.44. In the presence of NaCl, total disaggregation was observed at [DNA]/[BCD]=570+/-10 due to competition between Na(+) and the dye molecules for DNA electronegative binding sites.

Localization of dipyridamole molecules in ionic micelles: effect of micelle and drug charges

The localization of the coronary vasodilator dipyridamole (DIP) in cationic cetyltrimethylammonium chloride (CTAC), anionic sodium dodecylsulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and lysophosphatidylcholine (HPS and LPC) micelles was investigated using fluorescence quenching by quenchers with known localization in the micelle (TEMPO and 5-doxyl and 12-doxyl stearic acids). The use of fluorescence quenching jointly with fluorescence and 1H-NMR spectral measurements shows that DIP molecules in both protonated and nonprotonated forms are localized in micelles near the region which separates their polar and nonpolar parts, the polarizable heteroaromatic cycle of DIP being close to the polar part and the nonpolar substituents penetrating the hydrophobic interior of the micelle. The electrostatic interaction between the protonated DIP molecules and micelle charges either moves DIP into the micelle interior (for cationic and zwitterionic micelles) or draws it closer to the micelle surface (for anionic ones). Our results could be relevant to the mechanism of DIP action since many data indicate the interaction of the drug with cell membranes. The ability of DIP to localize near the membrane surface with the substituents immersed into a hydrophobic moiety could be essential for the drug interaction with P-glycoprotein, which is responsible for mediation of the effects of several antitumour drugs.

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.