Hidetake Imasato

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.

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.

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.

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.

Fluorescence Studies of Extracellular Hemoglobin of Glossoscolex paulistus in Met Form Obtained from Sephadex Gel Filtration

Abstract Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus in the met form presents an unique band at pH 7.0 and two bands at pH 9.0 as a result of alkaline dissociation. SDS-PAGE of the intact protein obtained at pH 7.0 is very similar to that for the oxyhemoglobin. Chromatography at pH 9.0 indicates complete dissociation of the oligomeric protein into two low molecular weight fractions corresponding to the trimers and monomers, showing that the oxidized extracellular hemoglobin is less stable than the oxyhemoglobin with respect to alkaline dissociation. Fluorescence quantum yields of different fractions obtained in the chromatography, as well as extinction coefficients at 280 nm and 415 nm, were estimated and compared to human methemoglobin. The fluorescence data are consistent with the high content of aromatic residues in G. paulistus hemoglobin. The increase in the fluorescence quantum yield upon both alkalinization and dissociation was correlated with the reduction of intramolecular quenching but the exposure of tryptophan residues to the solvent did not changed significantly as occurs for the oxy form. The intact native protein has a quantum yield of 0.36% at pH 7.0, increasing to 1.89% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 1.1% at pH 7.0 increasing to 1.43% at pH 9.0. The maximum emission wavelength of the intact protein (330 nm) is consistent with tryptophan residues being relatively buried; they become more fluorescent upon dissociation into smaller subunits but not more exposed since the wavelength of maximum emission is essentially unchanged at pH 9.0. In the monomer, the tryptophan residues also remain buried inside the protein molecule at pH 9.0 (328 nm). The dependencies of fluorescence quantum yields on the pH show in a clear way the hemichrome transitions observed by optical absorption spectroscopy indicating that the formation of two types of hemichromes accompany the distinct increase in fluorescence quantum yield. One type of hemichrome is irreversibly formed around pH 7.5–8.0 and a second reversible hemichrome is formed above pH 9.5–10.0. They are associated with the bis-imidazole low spin hemichrome and with a high spin hemichrome where the weakening of the iron bond to proximal histidine takes place. Addition of cyanide to the metHb solution produces the cyanomet form of the protein which is considerably more stable towards alkaline dissociation and presents a smaller change in quantum yield as a function of pH. Circular dichroism suggests that the formation of hemichromes is not accompanied by significant protein denaturation.

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.

Fluorescence properties of tryptophan residues in the monomeric d-chain of Glossoscolex paulistus hemoglobin: an interpretation based on a comparative molecular model.

The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C(varepsilon2) and C(delta2) atoms) for W15 and W129 were 16.8 and 18.5 A, respectively, from the geometric center of the heme, and W33 was located in close proximity to the heme group at a distance which was approximately half of that for W15 and W129. It was possible to identify three rotamers of W33 on the basis of electrostatic and Van der Waals energy criteria. The calculated distances from the center of the heme were 8.3, 8.4 and 9.1 A for Rot1, Rot2 and Rot3, respectively. Radiationless energy transfer from the excited indole to the heme was calculated on the basis of Förster theory. For W33, the distance was more important than the orientation factor, kappa(2), due to its proximity to the heme. However, based on kappa(2), Rot2 (kappa(2)=0.945) was more favorable for the energy transfer than Rot1 (kappa(2)=0.433) or Rot3 (kappa(2)=0.125). In contrast, despite its greater distance from the heme, the kappa(2) of W129 (2.903) established it as a candidate to be more efficiently quenched by the heme than W15 (kappa(2)=0.191). Although the Förster approach is powerful for the evaluation of the relative efficiency of quenching, it can only explain pico- and sub-nanosecond lifetimes. With the average lifetime, =3 ns, measured for the apomonomer as the reference, the lifetimes calculated for each emitter were: W33-1 (1 ps), W33-2 (2 ps), W33-3 (18 ps), W129 (100 ps), and W15 (600 ps). Experimentally, there are four components for oxymonomers at pH 7: two long ones of 4.6 and 2.1 ns, which contribute approximately 90% of the total fluorescence, one of 300 ps (4%), and the last one of 33 ps (7.4%). It is clear that the equilibrium structure resulting from homology modeling explains the sub-nanosecond fluorescence lifetimes, while the nanosecond range lifetimes require more information about the protein in solution, since there is a significant contribution of lifetimes that resemble the apo molecule.

Ionization and binding equilibria of papaverine in ionic micelles studied by 1H NMR and optical absorption spectroscopy

The binding of the vasodilator drug papaverine (PAV) to micelles of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS), cationic cetyltrimethylammonium chloride (CTAC) and anionic sodium dodecylsulfate (SDS) in aqueous solution was studied by 1H NMR and electronic absorption spectroscopy. In the presence of HPS or CTAC, the apparent pK(a) of PAV decreased by about 2 units, while it increased by about 2 units upon binding to SDS. However, the chemical shift patterns of both protonated (PAVH+) and deprotonated (PAV0) forms of PAV are not sensitive to the type of surfactant. The association constants were estimated as 5 +/- 2 M(-1) for PAVH+-CTAC, 8 +/- 3 M(-1) for PAVH+-HPS, (7 +/- 2) x 10(5) M(-1) for PAVH+-SDS, and 1.5 x 10(3) to 3.0 x 10(3) M(-1) for the complexes of PAV0 with all three types of micelles. Using these data, an electrostatic potential difference on the micelle-water interface was calculated as 150 +/- 10 mV for CTAC, 140 +/- 10 mV for HPS and - 140 +/- 10 mV for SDS. The results suggest that PAV aromatic rings are located in the hydrophobic part of the micelle. The electrostatic attraction or repulsion of the protonated quinoline nitrogen and surfactant headgroups changes the affinity of PAV to micelles and, thus, shifts the ionization equilibrium of PAV. The electrostatic potential of HPS micellar surface is determined by the cationic dimethylammonium headgroup fragment, whereas the anionic sulfate fragment attenuates the effective charge of HPS headgroup.