Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants.

Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.

Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin.

The application of an external electric field to dry films of Asp-85-->Asn mutant bacteriorhodopsin causes deprotonation of the Schiff base, resulting in a shift of the optical absorption maximum from 600 nm to 400 nm. This is in marked contrast to the case of wild-type bacteriorhodopsin films, in which electric fields produce a red-shifted product whose optical properties are similar to those of the acid-blue form of the protein. This difference is due to the much weaker binding of the Schiff-base proton in the mutant protein, as indicated by its low pK of approximately 9, as compared with the value pK approximately 13 in the wild type. Other bacteriorhodopsins with lowered Schiff-base pK values should also exhibit a field-induced shift in the protonation equilibrium of the Schiff base. We propose mechanisms to account for these observations.

Profluorescent protease substrates: intramolecular dimers described by the exciton model.

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.

Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

Photodissociation of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)-cobalt(III)] complex: a tool to study fast biological reactions involving O2.

Upon photolysis at 355 nm, dioxygen is released from a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)cobalt-(III)] complex in aqueous solutions and at physiological pH with a quantum yield of 0.04. The [Co(bpy)2(H2O)2]2+ (bpy = bipyridyl) photoproduct was generated on a nanosecond or faster time scale as determined by time-resolved optical absorption spectroscopy. A linear correspondence between the spectral changes and the oxygen production indicates that O2 is released on the same time scale. Oxyhemoglobin was formed from deoxyhemoglobin upon photodissociation of the (mu-peroxo) (mu-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex, verifying that dioxygen is a primary photoproduct. This complex and other related compounds provide a method to study fast biological reactions involving O2, such as the reduction of dioxygen to water by cytochrome oxidase.

Avaliação do efeito de diferentes concentrações de fotoiniciadores em propriedades mecânicas e na cor de um adesivo dental experimental

O presente trabalho teve por foco prover uma revisão de conceitos relacionados às resinas dentais fotopolimeráveis e, também, analisar como diferentes concentrações de fotoiniciadores de uso comum em odontologia podem influenciar no desempenho mecânico e na cor de resinas dentais. Concentrações de 0,01%, 0,10%, 0,25% 0,50%, 0,75%, 1,00% e 10% em massa de fotoiniciador foram adicionadas em uma matriz polimérica composta por TEGDMA/BisGMA 50%/50% em massa de forma que, fixando variáveis externas referentes à fonte de luz e à preparação das amostras, foi possível avaliar o efeito da concentração de fotoiniciador nas propriedades mecânicas de resistência à fratura por compressão, resistência à fratura pela aplicação de tensão diametral e dureza Barcol. Por fim, o efeito da concentração na cor aferida através do método CIELab foi investigada. Os fotoiniciadores utilizados foram o Irgacure® 819, a 1-fenil-1,2-propanodiona (PPD) e a Canforoquinona a qual foi utilizada juntamente com ocoiniciador Etil-4 dimetilaminobenzoato (EDB) na concentração constante de 1% em massa. Os resultados mecânicos e de cor foram comparados entre as diferentes amostras através de análises estatísticas (ANOVA) e, especificamente para cor, o parâmetro de diferença de cor (ΔE) foi interpretado verificar se as alterações observadas são perceptíveis ao olho humano. Para fundamentar os estudos, análises do espectro de absorção das amostras, do espectro de irradiação da fonte de luz e do grau de conversão das amostras foram realizadas. Os resultados revelaram que, independentemente do fotoiniciador, graus de conversão da mesma ordem de grandeza de um adesivo comercial foram obtidos, porém as velocidades de reação foram diferentes (maior velocidade para as amostras de CQ e menor para PPD). Curvas características foram obtidas para os espectros de absorção e de irradiação confirmando, pela sobreposição das curvas, que a fonte de luz é efetiva para iniciação da polimerização das amostras. As análises de resistência à fratura revelaram que a quebra catastrófica do material ocorrerá preferencialmente quando a força é aplicada ao longo do diâmetro para os corpos de prova preparados. Quanto à dureza, verificou-se que que para a CQ houve um máximo, para Irgacure um comportamento assintótico com aumento da concentração e, para a PPD, não foi possível a obtenção de amostras (verificado que a PPD necessita de tempo de irradiação superior ao convencionalmente utilizado na prática odontológica). Os testes de ANOVA permitiram verificar que o Irgacure é capaz de fornecer resultados mecânicos semelhante em relação à CQ ocasionando, em contrapartida, um menor escurecimento e amarelamento. Analisando o parâmetro ΔE, confirmou-se que a alteração na cor se torna evidente à medida que ocorre o aumento da concentração de fotoiniciador independentemente do fotoiniciador utilizado, porém a alteração é maior para a CQ.

Chiral, Three-Dimensional Supramolecular Compounds: Homo- and Bimetallic Oxalate- and 1,2-Dithiooxalate-Bridged Networks. A Structural and Photophysical Study.

In analogy to the [M(II)(bpy)(3)](2+) cations, where M(II) is a divalent transition-metal and bpy is 2,2'-bipyridine, the tris-chelated [M(III)(bpy)(3)](3+) cations, where M(III) is Cr(III) or Co(III), induce the crystallization of chiral, anionic three-dimensional (3D) coordination polymers of oxalate-bridged (&mgr;-ox) metal complexes with stoichiometries [M(II)(2)(ox)(3)](n)()(2)(n)()(-) or [M(I)M(III)(ox)(3)](n)()(2)(n)()(-). The tripositive charge is partially compensated by inclusion of additional complex anions like ClO(4)(-), BF(4)(-), or PF(6)(-) which are encapsulated in cubic shaped cavities formed by the bipyridine ligands of the cations. Thus, an elaborate structure of cationic and anionic species within a polymeric anionic network is realized. The compounds isolated and structurally characterized include [Cr(III)(bpy)(3)][ClO(4)] [NaCr(III)(ox)(3)] (1), [Cr(III)(bpy)(3)][ClO(4)][Mn(II)(2)(ox)(3)] (2), [Cr(III)(bpy)(3)][BF(4)] [Mn(II)(2)(ox)(3)] (3), [Co(III)(bpy)(3)][PF(6)][NaCr(III)(ox)(3)] (4). Crystal data: 1, cubic, P2(1)3, a = 15.523(4) Å, Z = 4; 2, cubic, P4(1)32, a = 15.564(3) Å, Z = 4; 3, cubic, P4(1)32, a = 15.553(3) Å, Z = 4; 4, cubic, P2(1)3, a = 15.515(3) Å, Z = 4. Furthermore, it seemed likely that 1,2-dithiooxalate (dto) could act as an alternative to the oxalate bridging ligand, and as a result the compound [Ni(II)(phen)(3)][NaCo(III)(dto)(3)].C(3)H(6)O (5) has successfully been isolated and structurally characterized. Crystal data: 5, orthorhombic, P2(1)2(1)2(1), a = 16.238(4) Å, b = 16.225(4) Å, c = 18.371(5) Å, Z = 4. In addition, the photophysical properties of compound 1 have been investigated in detail. In single crystal absorption spectra of [Cr(III)(bpy)(3)][ClO(4)][NaCr(III)(ox)(3)] (1), the spin-flip transitions of both the [Cr(bpy)(3)](3+) and the [Cr(ox)(3)](3)(-) chromophores are observed and can be clearly distinguished. Irradiating into the spin-allowed (4)A(2) --> (4)T(2) absorption band of [Cr(ox)(3)](3)(-) results in intense luminescence from the (2)E state of [Cr(bpy)(3)](3+) as a result of rapid energy transfer processes.

An ultraviolet multiplet table

Mode of access: Internet.

Embracing ligands. Synthesis, characterisation and the correlation between 59Co NMR and ligand field parameters of Co(III) complexes with a new class of nitrogen-thioether multidentate ligand

The syntheses of the hexadentate ligands 2,2,10,10-tetra(methyleneamine)-4,8-dithiaundecane (PrN(4)S(2)amp), 2,2,11,11-tetra(methyleneamine)-4,9-dithiadodecane (BuN(4)S(2)amp), and 1,2-bis(4,4-methyleneamine)-2-thiapentyl)benzene (XyN(4)S(2)amp) are reported and the complexes [Co(RN(4)S(2)amp)](3+) (R = Pr, Bu, Xy) characterised by single crystal X-ray study. The low-temperature (11 K) absorption spectra have been measured in Nafion films. From the observed positions of both spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(1g) and (1)A(1g) --> T-3(2g) bands, octahedral ligand-field parameters (10D(q), B and C) have been determined. DFT calculations suggest that significant interaction between the d-d and CT excitations occurs for the complexes. The calculations offer an explanation for the observed deviations from linearity of the relationship between Co-59 magnetogyric ratio and beta(DeltaE)(-1) (beta = the nephelauxetic ratio; DeltaE the energy of the (1)A(1g) --> T-1(1g) transition) for a series of amine and mixed amine/thioether donor complexes.

Synthesis and characterization of cobalt(III) complexes with sinambic amplector ligands

The synthesis of the hexadentate ligand 5,6-dimethyl-2,2,9,9-tetra(methyleneamine)-4,7-dithiadecane (1,2-Me(2)EtN(4)S(2)amp) is reported. The diastereiosomers were separated as cobalt(III) complexes using cation exchange chromatography. The rac and mesa isomers were characterized by NMR (C-13, H-1, Co-59), ESI-MS, UV-Vis spectroscopy and cyclic voltammetry. Single crystals of [Co(rac-1,2-Me(2)EtN(4)S(2)amp)] Cl-2(ClO4) (.) 2H(2)O were characterized by X-ray diffraction. The low-temperature (11 K) absorption spectra of the complexes have been measured in Nafion films and from the observed positions of both spin-allowed (1)A(1g) --> T-1(1g) and (1)A(1g) --> T-1(2g) and spin forbidden (1)A(1g) --> T-3(2g) bands, octahedral ligand-field parameters (10Dq, B and C) were determined. These results, in conjunction with the Co-59 NMR data, are used to further explore the relationship between the Co-59 magnetogyric ratio (gamma(Co)) and the product of the nephelauxetic ratio and the wavelength of the (1)A(1g) --> T-1(1g) transition (beta(DeltaE)(-1)) for complexes of mixed donor nitrogen-thioether ligands. (C) 2004 Elsevier Ltd. All rights reserved.

A PbS quantum-cube: conducting polymer composite for photovoltaic applications

We have developed a new non-polar synthesis for lead sulfide (PbS) quantum-cubes in the conjugated polymer poly-2-methoxy, 5-(2-ethyl-hexyloxy-p-phenylenevinylene) MEH-PPV. The conducting polymer acts to template and control the quantum-cube growth. Transmission electron microscopy of the composites has shown a bimodal distribution of cube sizes between 5 and 15 nm is produced with broad optical absorption from 300 to 650 nm. Photoluminescence suggests electronic coupling between the cubes and the conducting polymer matrix. The synthesis and initial characterization are presented in this paper. (C) 2003 Elsevier B.V. All rights reserved.

Time-resolved spectroscopy of the metal-to-metal charge transfer excited state in dinuclear cyano-bridged mixed-valence complexes

Visible pump-probe spectroscopy has been used to identify and characterize short-lived metal-to-metal charge transfer (MMCT) excited states in a group of cyano-bridged mixed-valence complexes of the formula [(LCoNCMII)-N-III(CN)(5)](-), where L is a pentadentate macrocyclic pentaamine (L-14) or triamine-dithiaether (L-14S) and M is Fe or Ru. Nanosecond pump-probe spectroscopy on frozen solutions of [(LCoNCFeII)-Co-14-N-III(CN)(5)](-) and [(LCoNCFeII)-Co-14S-N-III(CN)(5)](-) at 11 K enabled the construction of difference transient absorption spectra that featured a rise in absorbance in the region of 350-400 nm consistent with the generation of the ferricyanide chromophore of the photoexcited complex. The MMCT excited state of the Ru analogue [(LCoNCRuII)-Co-14-N-III(CN)(5)](-) was too short-lived to allow its detection. Femtosecond pump-probe spectroscopy on aqueous solutions of [(LCoNCFeII)-Co-14-N-III(CN)(5)](-) and [(LCoNCFeII)-Co-14S-N-III(CN)(5)](-) at room temperature enabled the lifetimes of their Co-II-Fe-III MMCT excited states to be determined as 0.8 and 1.3 ps, respectively.

Towards structure-property-function relationships for eumelanin

We discuss recent progress towards the establishment of important structure-property-function relationships in eumelanins-key functional bio-macromolecular systems responsible for photoprotection and immune response in humans, and implicated in the development of melanoma skin cancer. We focus on the link between eumelanin's secondary structure and optical properties such as broad band UV-visible absorption and strong non-radiative relaxation; both key features of the photo-protective function. We emphasise the insights gained through a holistic approach combining optical spectroscopy with first principles quantum chemical calculations, and advance the hypothesis that the robust functionality characteristic of eumelanin is related to extreme chemical and structural disorder at the secondary level. This inherent disorder is a low cost natural resource, and it is interesting to speculate as to whether it may play a role in other functional bio-macromolecular systems.

Chemical and structural disorder in eumelanins: A possible explanation for broadband absorbance

We report the results of an experimental and theoretical study of the electronic and structural properties of a key eumelanin precursor-5,6,-dihydroxyindole-2-carboxylic acid ( DHICA) - and its dimeric forms. We have used optical spectroscopy to follow the oxidative polymerization of DHICA to eumelanin and observe red shifting and broadening of the absorption spectrum as the reaction proceeds. First principles density functional theory calculations indicate that DHICA oligomers ( possible reaction products of oxidative polymerization) have the gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital red-shifted gaps with respect to the monomer. Furthermore, different bonding configurations ( leading to oligomers with different structures) produce a range of gaps. These experimental and theoretical results lend support to the chemical disorder model where the broadband monotonic absorption characteristic of all melanins is a consequence of the superposition of a large number of nonhomogeneously broadened Gaussian transitions associated with each of the components of a melanin ensemble. These results suggest that the traditional model of eumelanin as an amorphous organic semiconductor is not required to explain its optical properties and should be thoroughly reexamined. These results have significant implications for our understanding of the physics, chemistry, and biological function of these important biological macromolecules. Indeed, one may speculate that the robust functionality of melanins in vitro is a direct consequence of its heterogeneity, i.e., chemical disorder is a "low cost" natural resource in these systems