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Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15mgkg(-1). Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000cps for 5mgkg(-1), 6h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7min) detectable only after injection of at least 15mgkg(-1). Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination.

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The interaction of cytochrome c with micelles of sodium dodecyl sulfate was studied by proton NMR spectroscopy. The protein/micelles ratio was found to be crucial in controlling the extent of the conformational changes in the heme crevice. Over a range of ratios between 1:30 and 1:60, the NMR spectra of the ferric form display no paramagnetic signals due to a moderately fast exchange between intermediate species on the NMR time scale. This is consistent with an interconversion of bis-histidine derivatives (His18-Fe-His26 and His18-Fe-His33). Further addition of micelles induces a high-spin species that is proposed to involve pentacoordinated iron. The resulting free binding site, also encountered in the ferrous form, is used to complex exogenous ligands such as cyanide or carbon monoxide. Attribution of the heme methyls was performed by means of exchange spectroscopy through ligand exchange or electron transfer. The heme methyl shift pattern of the micellar cyanocytochrome in the ferric low spin form is different from the pattern of both the native and the cyanide cytochrome c adduct, in the absence of micelles, reflecting a complete change of the heme electronic structure. Analysis of the electron self-exchange reaction between the two redox states of the micellar cyanocytochrome c yields a rate constant of 2.4 x 10(4) M(-1) s(-1) at 298 K, which is surprisingly close to the value observed in the native protein.

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This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.

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The geometry about the Ru atom in the title compound, [Ru(C(9)H(9)N)(2)(C(92)H(108)N(4)O(24))], is approximately tetragonal and the porphyrin ring is nearly planar, while the C-N-R angles [169.3 (3) and 163.9 (3) degrees ] of the isocyanide ligands in the complex are different from the value of 180 degrees expected in the free ligand.

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An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.

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A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s.

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The 1H-NMR study of diamagnetic cytochrome P450cam FeII-CO has been performed for the first time. Chemical shifts of the cysteinate fifth ligand protons and of several heme protons have been assigned through 1- and 2-dimensional spectra at 500 MHz. A substrate dependance has been observed for the resonance of the cysteinate proton detected in the high-field region.

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For the first time, Fourier transform infrared spectroscopy has been applied to cytochrome P-450 to analyze the protein secondary structure. From Fourier self-deconvolution and fitting the infrared spectra in the amide I' region (1600-1700 cm-1), we estimate 44% alpha-helix, 31% beta-sheet, and 18% turns for substrate-free cytochrome P-450cam. In the presence of camphor, 54% alpha-helix and 310-helix, 21% beta-sheet, and 21% turns are obtained which agree with the crystallographic data of 53% alpha-helix, 19% beta-sheet, and 16% turns [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. Cytochrome P-420cam is produced from substrate-free cytochrome P-450cam in two ways: (i) by temperature elevation up to 60 degrees C and (ii) by exposure to KSCN up to 1.5 M. The secondary structure composition is determined for each temperature and KSCN concentration and compared with the changes observed in the iron ligand CO stretch vibration bands appearing between 1900 and 2000 cm-1. Thermally induced cytochrome P-420 has an alpha-helix content of 19%, a beta-sheet content of 53%, 14% turns, and 5% antiparallel beta-sheets from intermolecular hydrogen bonds within protein aggregates. The formation of cytochrome P-420 as a function of the KSCN concentration indicates two types of cytochrome P-420. Up to 1 M KSCN, the induced cytochrome P-420 displays only little modification of the secondary structure, whereas at 1.5 M KSCN, larger changes are observed, resulting in 85% cytochrome P-420 without protein precipitation and containing 30% alpha-helix, 48% beta-sheet, and 17% turns. Infrared spectra in the iron ligand CO stretch region show several subconformers for cytochrome P-420. During the cytochrome P-420 formation, the CO stretch modes are shifted to higher frequencies by 3-11 cm-1, with a main feature at about 1964 cm-1, compared to those of substrate-free cytochrome P-450cam-CO.

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13CO NMR chemical shifts and 12CO infrared stretching frequencies have been measured for cytochrome P-450cam-CO in the presence of D-camphor and of various camphor analogues. A linear correlation between both parameters delta (13C) and v(CO) was found indicating that the steric and electrostatic interactions acting on the CO ligand are influenced by the substrate. It has been proved that P-450 complexes are on another line in this correlation than hemoglobins which is explained by the different proximal ligand.

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Two-dimensional nuclear magnetic resonance techniques have been used to assign resonances corresponding to the heme pocket and several other residues of horse heart and sperm whale myoglobins ligated by trimethylphosphine. The assignment procedure was based mainly on the nuclear Overhauser effect connectivities with the ligand and the heme substituents. For quantitative measurements of Overhauser effects, application of truncated driven techniques between a proton from distal residues and methyl groups from the ligand was used to determine internuclear distances. These new results have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between horse heart and sperm whale myoglobins. The interproton distances between distal amino acid residues and trimethylphosphine were found to be longer in horse heart myoglobin relative to those in sperm whale myoglobin. This result suggests that the size of the heme pocket is larger in horse heart myoglobin. Association and dissociation rate constants were measured for trimethylphosphine binding to myoglobins. Both values were four times larger for horse heart myoglobin than those for sperm whale myoglobin. This observation confirms the structural results obtained with NMR studies and is rationalized by a greater stabilization of a larger pocket in horse heart myoglobin relative to sperm whale myoglobin.

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Tetra-p-aminophenylporphyrin (TPP) was conjugated with gadolinium diethylenetriaminepentaacetic acid (DTPA) and used as a contrast agent in magnetic resonance (MR) imaging to achieve tumor selectivity in nude mice. A substantial decrease in T1 was measured in excised tissues (kidneys, tumor, and liver) from mice that received the porphyrin derivative Gd2(DTPA)4 TPP. Toxicity and phototoxicity were less than those obtained with hematoporphyrin derivative in both L1210 lymphoblastic leukemia cells and HT 29 human colonic cancer cells, as determined with in vitro assays. MR images showed an enhancement of contrast between the tumor and adjacent tissue after injection of this agent. The results indicate that Gd2(DTPA)4TPP could be a useful prototype paramagnetic porphyrin MR imaging contrast agent with an affinity for tumors.

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Electron self-exchange has been measured by an NMR technique for horse-heart myoglobin. The rate is 3.1 x 10(3) M-1 s-1 at 23 degrees in 0.1 M phosphate at pH 6.9. The rate was weakly dependent on ionic strength up to 0.7 M in added KCl (3.9 x 10(3) M-1 s-1). The enthalpy of activation was 12.1 +/- 0.5 kcal mol-1, and the entropy of activation was -1.2 +/- 0.5 cal mol-1 deg-1. Analysis of the data in terms of the Marcus theory gives a reorganization energy, lambda, for self-exchange of 1.6 eV mol-1.

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1H-NMR techniques have been used to study the metal binding properties of a synthetic peptide of 15 amino acids corresponding to a highly conserved domain of Pleurodeles lectin. The addition of lanthanum chloride or praseodymium chloride in a peptide solution induces some conformational changes as displayed by several concerted variations of peptide resonances. The Ln3+ concentration dependence of the chemical shifts was used to calculate the Ln3+ binding constants. The dissociation constants of 95 microM and 280 microM were found for La3+ and Pr3+, respectively.

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31P-NMR of trimethylphosphine binding to the ferrous chains of a ([alpha Fe(II), beta Mn(II)]hemoglobin hybrid is employed to investigate partially liganded species. This study shows that at low pH (6.5), in the presence of inositol hexaphosphate, the resonance at 23.2 ppm (from H3PO4) is due to phosphine bonding to alpha-chains in the T quaternary state. At elevated pH (7.6), phosphine binding to the alpha-chains produces a resonance at 24.8 ppm which is associated with a T-to-R conversion. These findings are discussed in relation with our previous results on direct observation of intermediate ligation states of hemoglobin.

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1H-NMR saturation transfer spectroscopy and nuclear Overhauser effect (NOE) have been utilized to assign several heme resonances in the low-spin trimethyl phosphine complex of sperm whale metmyoglobin. The two methods permit the location of the heme methyl resonances and the heme 2-vinyl group resonances. A qualitative comparison involving the methyl shift pattern in metMbN3, metMbCN, imidazole metMb and trimethyl phosphine metMb shows a reverse methyl shift between pyrrole I and pyrrole IV. The different hyperfine shift pattern for metMbPMe3 is suggested to arise from: (i) a possible reorientation of the proximal histidine plane; (ii) different heme protein contacts in the different ligated proteins, and (iii) a small contribution from high-spin character. The 2-vinyl group is formed in the cis plane orientation.

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Binding of trimethylphosphine to myoglobins and hemoglobins from a variety of sources has been examined by 1H-nuclear magnetic resonance. The hemoglobins exhibit two resonances at high field (approx. -3.5 ppm) which have been assigned to PMe3 bound to alpha or to beta subunits. Perturbations in the beta heme pocket induced by a thiol reagent have been detected both in 1H and 31P spectra.

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A 31P-NMR study of trimethyl phosphine bound to the heme iron(II) atom in natural myoglobins, hemoglobins and synthetic porphyrin iron(II) shows that the iron-bound trimethylphosphine 31P chemical shifts are sensitive to the presence of globin:separated NMR signals can be observed for 31PMe3 bound to the hemes of the alpha and beta chains. On the basis of previous hemoprotein studies, the markedly high field resonance observed with one of the two PMe3-rabbit subunits is consistent with a specific role of the distal histidine (E7) in rabbit alpha-subunit.

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