Characterization of a human and mouse tetrapyrrole-binding protein

The cDNA for p22HBP has been cloned from human and mouse, and the protein expressed, purified, and characterized. Both mouse and human proteins bind heme and porphyrins with micromolar K(d)s, are highly homologous, monomeric, and soluble, and have a cytoplasmic location. The proteins bind metalloporphyrins, free porphyrins, and N-methylprotoporphyrin with similar affinities, and mutations of a selected set of putative metal ligating residues did not have any significant effect on the measured K(d)s. That the presence or absence of metal in the porphyrin has no effect on the binding constants and the observation that the EPR signal for heme does not change upon binding to the protein strongly suggest that p22HBP is a generic tetrapyrrole-binding protein rather than a dedicated heme-binding protein. A role for p22HBP in cellular porphyrin metabolism is discussed.     

Binding of octa-plus porphyrazines to DNA

We present a preliminary report of the interaction of octacationic N-methyl-pyridyl metalloporphyrazines, M=Cu(II) (Cupz⁺⁸) and Zn(II) (Znpz⁺⁸), with calf thymus DNA. These were monitored by electronic absorption spectroscopy, and in the case of Znpz⁺⁸, also by emission spectroscopy. These studies show that both the Cupz⁺⁸ and Znpz⁺⁸ interact strongly with DNA, and at sufficient concentrations induce a highly colored precipitate. The spectrum of Cupz⁺⁸ red-shifts as DNA is added, which is interpreted as indicative of π stacking of the charged macrocycle as it binds electrostatically to the outside of the DNA duplex. The spectrum of Znpz⁺⁸ does not shift, indicative of simple electrostatic surface binding. The absence of stacking may be related to the presence of an axial metal-ion ligand for Znpz⁺⁸, presumably 1120, while the metal ion of Cupz⁺⁸ is four-coordinate.     

A simple method for screening porphyrin secretion by colonies of Escherichia coli

Abstract Escherichia coli secretes porphyrins when exposed to 20 mM 1-thioglycerol. A method was devised to isolate mutants, which do not excrete porphyrins in the presence of thiols. DEAE-Sephadex beads were incorporated into plates growing colonies of E. coli . The secreted porphyrins on these plates formed a fluorescent halo around each colony, when the plates were viewed under long-wave ultraviolet light. Mutants were found, that formed either halo-less colonies or a colony with excess halo. This method of incorporating immobilized ion exchangers into plates may be useful in isolating non-secretor or super-secretor mutants of a variety of organisms.     

在光作用下卟啉和蛋白复合物的发光特性——癌固有荧光发光物质的探讨

本文研究了在光作用下,原卟啉和白蛋白复合物的发光特性,讨论了这些特性与临床诊断中所选择的癌固有荧光峰的关系。     

弱激光对改善血红蛋白携氧功能的机理研究

本文用群论分析了卟啉的对称结构,并用量子力学导出激光－铁卟啉相互作用系统的自由能,改进了文献「２」的工作,对激光血管内照射能增强血液携氧能力的现象进行诠释。     

Improvement of porphyrins for G-quadruplex DNA targeting

G-quadruplex nucleic acids are emerging as therapeutic targets for small molecules referred to as small-molecule G-quadruplex ligands. The porphyrin H₂-TMPyP4 was early reported to be a suitable motif for G-quadruplex DNA recognition. It probably binds to G-quadruplex nucleic acid through π-π stacking with the external G-quartets. We explored chemical modifications of this porphyrin such as insertion of various metal ions in the centre of the aromatic core and addition of bulky substituents that may improve the specificity of the compound toward G-quadruplex DNA. Porphyrin metallation, affording a G4-ligand with two symmetric faces, allowed the conclusion that the presence of an axial water molecule perpendicular to the aromatic plane lowered but did not hamper π-π stacking interactions between the aromatic parts of the ligand on the one hand and the external G-quartet on the other. The charge introduced in the centre of the porphyrin had little influence on binding. Thus, the ionic channel in the centre of G-quadruplex nucleic acids was not found to provide clear additional molecular clues for G-quadruplex nucleic acids targeting by porphyrins tested in the present study. Furthermore, we confirmed the unique G-quadruplex selectivity of a porphyrin modified with four bulky substituents at the meso positions and showed that although the compound is not “drug-like” it was capable of entering cells in culture and mediated some of the typical cellular effects of small-molecule G-quadruplex ligands.     

Enhancers of the non-peroxidative metal porphyrin chemiluminescence reaction

Metal porphyrins catalyse luminol chemiluminescence at pH 13 without added peroxide. The effects of 22 different surface active compounds on this reaction were studied using six metal porphyrins and one metal porphyrin conjugate. The most active catalyst was Mn-meso-tetra(4-sulphonatophenyl)porphine. Tween-20 enhanced the activity of this catalyst best at a Tween-20 to luminol ratio of 74:1. However, lauryl sulphate enhanced best at an optimum lauryl sulphate to luminol ratio of over 1000:1 and both detergents enhanced the reaction when present below their critical micelle concentrations. Negatively charged aliphatic compounds such as fatty acids enhanced the reaction but positive-charged aliphatic compounds inhibited it. Small differences in enhancer structure resulted in differing enhancement. For example, linoleic acid enhanced Mn-meso-tetraphenyl porphine more than 10-fold, yet linolenic acid inhibited this catalyst. Conjugation of a metal porphyrin to antibody did not influence its enhancement by detergents. The results indicate that the enhancement mechanism does not require formation of pure detergent micelles but that direct association between enhancer and catalyst may be important.     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Coordination of bismuth and lead in porphyrins: Towards an in-situ generator for α-radiotherapy?

In order to investigate the possible coordination of both bismuth(III) and lead(II) by porphyrins for an application in alpha-radioimmunotherapy, several series of functionalized bismuth porphyrins have been synthesized and their stabilities in acidic medium were compared.Where unfunctionalized porphyrins are not suitable for such a purpose, strapped hanging carboxylate porphyrins are stable and allow a rapid coordination of both bismuth and lead. This property could lead to the preparation of an in-situ generator of alpha particles by a pre-complexation of ²¹²lead, the radioactive parent of ²¹²bismuth, in the ligand.     

Effects of porphyrins and inorganic iron on the growth of Prevotella intermedia

We demonstrated earlier that hemin-iron-containing compounds which include hemin, human hemoglobin, bovine hemoglobin, and bovine catalase stimulate the growth of Prevotella intermedia [Leung, Subramaniam, Okamoto, Fukushima, Lai, FEMS Microbiol. Lett. 162 (1998) 227-233]. However, the contributions of tetrapyrrole porphyrin ring in these hemin-iron sources as well as inorganic iron for the growth of this organism have not been determined. The purpose of this study was to examine the effects of porphyrins, host iron-binding proteins, and various inorganic iron sources on the growth of hemin-iron depleted P. intermedia. Protoporphyrin IX and protoporphyrin IX-zinc, either in the presence or absence of supplemented ferrous or ferric iron, promoted the growth of P. intermedia at a rate that was comparable to that of the hemin control. On the other hand, neither the host iron proteins, transferrin and lactoferrin, nor the inorganic iron sources which included ferrous chloride, ferric chloride, ferric citrate, ferric nitrate, and ferric ammonium citrate at concentrations up to 200 microM stimulated the growth of hemin-iron-restricted P. intermedia. The results suggest that P. intermedia only use iron in a specific form and that the porphyrin-ring structure is essential for the growth of P. intermedia as in the case of other related organisms.     

Synthesis, characterization, and distortion properties of vanadyl complexes of octaphenylporphyrin and dodecaphenylporphyrin

In order to investigate the influence of ligand distortion on metal centers of porphyrin complexes, distorted vanadyl porphyrin complexes, VO(OPP) (OPP = 2,3,5,10,12,13,15,20-octaphenylporphinato) and VO(DPP) (DPP = 2,3,5,7,8,10,12,13,15,17,18,20-dodecaphenylporphinato), have been prepared. In the crystal structures, VO(OPP) and VO(DPP) had a ruffled structure and a saddle-shaped structure, respectively. In addition, these complexes exhibited red shift and broadening of the absorption bands in the UV-Vis spectra and significant negative shifts of oxidation potentials of the porphyrin ligands in the cyclic voltammograms compared with those of the planar VO(TPP) (TPP = tetraphenylporphinato). These results indicate that the OPP and DPP complexes have the distorted structures both in solids and in solutions. The VO bond characters of VO(TPP), VO(OPP), and VO(DPP) do not show the significant difference in their crystal structures and resonance Raman spectra. This suggests that the distortion of porphyrin ligand little affects the Lewis acidity of the metal center. The non-planar porphyrin distortion gives the change of HOMO-LUMO gap.     

Resonance Raman investigation of lysine and N-acetylmethionine complexes of ferric and ferrous microperoxidase

In order to evaluate the steric and electronic effects of mixed axial ligations on the heme c structure, lysine (Lys) and N-acetylmethionine (AcMet) complexes of ferric and ferrous microperoxidase-8 (MP8(III) and MP8(II), respectively) are characterized by absorption and resonance Raman (RR) spectroscopies. Spectrophotometric titrations establish that MP8(III) binds one molecule of exogenous ligand while MP8(II) forms mono(ligated) and bis(ligated) compounds. The Soret-excited RR spectra of the six-coordinated low-spin MP8(III) complexes show that the macrocycle can adopt different structures between planar and ruffled conformations. The ferriheme c conformation is primarily determined by the ionization state of the His side chain of MP8(III) and, secondarily, by the bonding and nonbonding heme-ligand interactions. As far as the RR spectra of the MP8(II) complexes are concerned, they permit us to conclude that the mixed His/Lys and His/AcMet coordinations induce a nonplanar heme conformation, the extent of deformation again depending on the ionization state of the endogenous His ligand. In contrast, the RR spectra of the bis(Lys) and bis(AcMet) compounds are associated with a planar heme structure. When the His of MP8 is bound to heme c, the stabilization of distorted heme conformations is thus associated with constraints exerted by the Cys-Ala-Gln-Cys-His-peptide on the porphyrin macrocycle. More generally, the spectroscopic data obtained in this study can be used to predict both the axial coordination and the structure of heme in c-type cytochromes. Received: 19 January 1998 / Revised version: 23 March 1998 / Accepted: 27 March 1998     

Regulation of responsiveness of phosphorescence toward dissolved oxygen concentration by modulating polymer contents in organic–inorganic hybrid materials

Platinum(II) octaethylporphyrin (PtOEP)-loaded organic–inorganic hybrids were obtained via the microwave-assisted sol–gel condensation with methyltrimethoxysilane and poly(vinylpyrrolidone). From transparent and homogeneous hybrid films, the strong phosphorescence from PtOEP was observed. Next, the resulting hybrids were immersed in the aqueous buffer, and the emission intensity was monitored by changing the dissolved oxygen level in the buffer. When the hybrid with relatively-higher amount of the silica element, the strong phosphorescence was observed even under the aerobic conditions. In contrast, the emission from the hybrids with lower amounts of the silica element was quenched under the hypoxic conditions. This is, to the best of our knowledge, the first example to demonstrate that the responsiveness of the phosphorescence intensity of PtOEP in hybrid films to the dissolved oxygen concentration in water can be modulated by changing the percentage of the contents in the material.     

Microcalorimetric and spectroscopic investigation of the antibacterial properties of cationic ytterbium(III)-porphyrin complexes lacking charged peripheral groups

The antibacterial activities towards Escherichia coli of two cationic Yb(III)-monoporphyrin complexes, [Yb(III)(TMP)(H2O)3]Cl (1) and [Yb(III)(TTP)(H2O)3]Cl (2), were investigated at the cellular and sub-cellular levels. The biological effects of the complexes on the growth of E. coli were evaluated by microcalorimetry and by analysis of the resulting metabolic thermogenic curves, from which IC50 values and metabolic parameters such as growth rate and generation time were derived. At the subcellular level, DNA-binding experiments were performed by means of UV/VIS- and fluorescence-titration experiments, as well as by near-infrared (NIR) emission, which revealed that 1 and 2 strongly bind to herring-sperm DNA (HS-DNA), though by different binding modes.     

Synthesis, and DNA-binding and DNA-photocleavage properties of multiply charged porphyrins

Four new cationic porphyrins, compounds 1-4, with five to seven positive charges, were synthesized, characterized, and investigated for their binding properties towards calf-thymus DNA (CT-DNA). UV/VIS and fluorescence-titration data indicated strong binding, the apparent binding constants (K(app); (1.3-10)x10(-6) M) increasing with increasing number of charges, as determined by competitive fluorescence titration using ethidium bromide (EB) as molecular probe. These results were qualitatively confirmed by the observed photocleavage efficiency of the porphyrins towards plasmid pBR322 DNA.     

Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities

A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exhibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application.     

Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins. Received: 28 January 1997 / Accepted: 13 March 1997     

Synthesis under solvent free conditions and photoluminescence study of ionic intermediate sitting-atop complexes of meso-tetraarylporphyrins and phosphorus oxychloride

The reaction of meso-tetraarylporphyrins with phosphorus oxychloride was studied. The reaction product is the so-called intermediate sitting-atop (i-SAT) complex where two pyrrolic nitrogen atoms of the porphyrin core coordinate to the phosphorus atom and two protons on the pyrrolic nitrogen atoms remain. Selection of solvent free conditions is caused that the reaction does not progress until deprotonation step of porphyrin and stopped in the intermediate step for formation of the sitting-atop complex, [POCl₂(H₂t(X)pp)]Cl. The sitting-atop complexes were characterized by (¹H, ³¹P, ¹³C) NMR, FT-IR, UV-vis and photoluminescence spectroscopy (PL), elemental analysis and electrical conductometry. Photoluminescence study of the complexes indicates that their emission spectra are different from those of free base porphyrins.     

Electronic structures of azulene-fused porphyrins as seen by magnetic circular dichroism and TD-DFT calculations

A combination of magnetic circular dichroism (MCD), electronic absorption spectroscopy and time-dependent density functional theory (TD-DFT) calculations has been used to investigate the electronic structure of azulene-fused pi-expanded porphyrins based primarily on the spectral properties of absorption bands in the near infrared region. From MCD experiments, it was suggested that in the case of a mono-azulene-fused porphyrin DeltaHOMO approximately equal DeltaLUMO (where DeltaHOMO is the magnitude of the energy gap between the HOMO and HOMO-1 and DeltaLUMO is the magnitude of the energy gap between the LUMO and LUMO+1), while in the case of an oppositely-di-azulene-fused porphyrin, DeltaHOMO相似文献