Tania T. Tominaga

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.

STATE-SPACE APPROACH FOR THE ANALYSIS OF SOIL WATER CONTENT AND TEMPERATURE IN A SUGARCANE CROP

The state-space approach is used to describe surface soil water content and temperature behaviour, in a field experiment in which sugarcane is submitted to different management practices. The treatments consisted of harvest trash mulching, bare soil, and burned trash, all three in a ratoon crop, after first cane harvest. One transect of 84 points was sampled, meter by meter, covering all treatments and borders. The state-space approach is described in detail and the results show that soil water contents measured along the transect could successfully be estimated from water content and temperature observations made at the first neighbour.

1H NMR AND ELECTRONIC ABSORPTION SPECTROSCOPY OF PARAMAGNETIC WATER-SOLUBLE MESO-TETRAARYLSUBSTITUTED CATIONIC AND ANIONIC METALLOPORPHYRINS

The ionization, mu-oxo-dimerization and axial ligation equilibria of free bases, iron(III) and manganese(III) derivatives of meso-tetrakis(p-sulfonatophenyl)porphyrin (TPPS4) and meso-tetrakis(4-N-methyl-pyridiniumyl)porphyrin (TMPyP) in aqueous solution are studied by 1H NMR and electronic absorption spectroscopy. At physiological pH, Fe(III) complexes of TMPyP and TPPS4 exist predominantly as dimers and may undergo transition to low spin species upon binding to biomolecules, whereas Mn(III) complexes are essentially monomeric. Dicyano and bis-imidazole complexes of FeTMPyP and FeTPPS4 are low spin monomer adducts in the pH range 2.0 to 11.2. No low spin dimeric complexes were found. The low spin monocyano and high spin mono-imidazole complexes of FeTMPyP are formed in acidic and alkaline media, respectively. T1-relaxation enhancement of water protons at 200 MHz induced by FeTPPS4 falls dramatically in the sequence high spin >> dimeric > low spin form.

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.

Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus.

Sephadex G-200 chromatography of the extracellular hemoglobin from the giant earthworm G. paulistus in the met form presents a single peak at pH 7.0 and two peaks at pH 9.0 as a result of alkaline dissociation. SDS-PAGE shows that the polypeptide chains are very similar to those observed for the oxy form and the two peaks at pH 9.0 correspond to the trimer contaminated by linkers and monomers which seems to be quite pure. The aquomet acid form is stable as an oligomer of molecular mass 3.1 x 10(6) Da only in a narrow pH range around neutrality. Increasing the pH above 7.5 leads to an irreversible transition from aquomet to hemichrome I which is the low-spin bis-imidazole complex. At pHs above 9.5-10.0 a second reversible transition takes place from hemichrome I to hemichrome II, a high-spin complex which is associated with the weakening and possible disruption of the proximal Fe--N histidine bond. Thus, increase in pH above 8.0 induces changes in the heme pocket that involve both the distal and proximal sides of the heme. EPR measurements show a very sharp decrease of the aquomet high-spin signal in the range of pH 7.0-8.0 and a very small low-spin signal even at liquid helium temperatures. The transition to hemichrome I is also accompanied by the loss of heme optical activity monitored by CD, which is consistent with the weakening of heme--globin interaction. Hemichrome I in the presence of cyanide gives the typical cyanometHb derivative which has a transition to a hemichrome at much higher pHs. This observation suggests that the dissociation of the oligomer in alkaline medium as well as the stability of the heme on the proximal side, depend both upon the ligand present at the sixth coordination position on the distal side. Hence, we believe that hemi(hemo)chrome formation in G. paulistus Hb and other invertebrate hemoglobins is a common phenomenon, not associated with protein denaturation, which may provide a fine tuning mechanism to control subunit interactions through changes in the distal side of the heme pocket.

Interaction of tyrosine and tyrosine dipeptides with Cu2+ ions: A fluorescence study

Abstract The interaction of tyrosine (Tyr) and the dipeptides glycyltyrosine (Gly-Tyr) and tyrosylglycine (Tyr-Gly) with copper ions has been studied through the use of fluorescence quenching due to the binding of the paramagnetic metal to the fluorophores. The pKa values for the protonation of the tyrosine derivatives were estimated from fluorescence data being 2.30 and 10.28 for Tyr, 3.00, 8.44 and 10.70 for Gly-Tyr and 2.98, 8.00 and 10.51 for Tyr-Gly. In the case of the pure amino acid the pKa of carboxylate and phenolic hydroxy groups were resolved, while for the dipeptides the pK for the amino group was additionally obtained. A simple equilibrium binding model allowed estimates of the association constants of 10−7 M−1 to be obtained for the formation of the complexes of dipeptides with copper. Stoichiometry of binding was 1:1 for the dipeptide-metal complex. In the case of Gly-Tyr and acidic pH values quenching of fluorescence was due to complexation of the carboxylate group with Cu2+ while for Tyr-Gly some quenching involving other sites was also observed. Additional ESR experiments suggested the involvement of the peptide and amino nitrogens in complex formation. Our data are in agreement with recent X-ray data for the crystal structure of Gly-Tyr-Cu2+ complex [I. Dey et al., J. Cryst. Spect. Res., 23 (1993) 1]. The metal is coordinated to amino and amide peptide nitrogens, carboxylate and to an oxygen from a water molecule.