Cyclic endoperoxides of β-carotene, potential pro-oxidants, as products of chemical quenching of singlet oxygen

AbstractPhotoprotection by carotenoids is generally considered to be based on the photophysical quenching of triplets and singlet oxygen. There is also accumulating evidence of an alternative, chemical quenching of triplets and singlet oxygen by carotenoids. We report the identification of relatively stable cyclic mono- and diendoperoxides as first products of such an alternative reaction. Nevertheless, these species remain reactive and in the dark cause autooxidation of β-carotene in our model system. Their formation could explain the intriguing pro-oxidant and cytotoxic activity of carotenoids

Antioxidant effects of carotenoids in a model pigment-protein complex

The effect of carotenoids on stability of model photosynthetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricyanide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carotenoids may significantly enhance the stability of photosynthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxidant

Controlling structural and functional features of photosynthetic antenna

In order to gain control over the assembly and functioning of photosynthetic antenna, we have developed methods to manipulate pigment composition of bacterial LH1 complexes via their reconstitution with modified bacteriochlorophylls or carotenoids, major photoactive cofactors of these antennae. In the present work we show how the reconstitution and pigment exchange approach is applied to control structural and functional parameters of LH1 and its subunits. The size of the subunits and the energy of the first excited singlet state can be controlled via the use of detergent while the thermodynamics of LH1 formation can be modified using carotenoids and/or a co-solvent. Carotenoids affect the efficiency of the intracomplex energy transfer, while the replacement of native bacteriochlorophyll a with its Ni-substituted analog allows one to control the excited state properties of LH1. These results show that LH1 is a very promising model system applicable for the design of bio-inspired device performing solar energy conversion

Ligation of water to magnesium chelates of biological importance

Water binding to several Mg^{2+} chelates, ethylenediamine, ethylenediamine-N,N’-diacetate, porphyrin, chlorophyll a and bacteriochlorophyll a, to form five- and six-coordinate complexes is studied by means of density functional theory. The results obtained for magnesium chelates are compared with the properties of the respective aqua complexes and the influence of the permittivity of environment on the ligand binding energies is discussed. Although the most common coordination number of Mg^{2+} is six, in the tetrapyrrolic chelates it is reduced to five because the accommodation of the sixth water ligand results in no gain in energy. This is in line with the experimental observations made for coordination of chlorophylls in vivo. The binding between Mg^{2+} and water is mostly of electrostatic nature, which is supported by the finding that its energy is correlated both with the electron density of the chelator and with electrostatic potential determined at the ligand binding site

Tuning the photophysical features of self-assembling photoactive polypeptides for light-harvesting

The LH1 complex is the major light-harvesting antenna of purple photosynthetic bacteria. Its role is to capture photons, and then store them and transfer the excitation energy to the photosynthetic reaction center. The structure of LH1 is modular and it cooperatively self-assembles from the subunits composed of short transmembrane polypeptides that reversibly bind the photoactive cofactors: bacteriochlorophyll and carotenoid. LH1 assembly, the intra-complex interactions and the light-harvesting features of LH1 can be controlled in micellar media by varying the surfactant concentration and by adding carotenoid and/or a co-solvent. By exploiting this approach, we can manipulate the size of the assembly, the intensity of light absorption, and the energy and lifetime of its first excited singlet state. For instance, via the introduction of Ni-substituted bacteriochlorophyll into LH1, the lifetime of this electronic state of the antenna can be shortened by almost three orders of magnitude. On the other hand, via the exchange of carotenoid, light absorption in the visible range can be tuned. These results show how in a relatively simple self-assembling pigment-polypeptide system a sophisticated functional tuning can be achieved and thus they provide guidelines for the construction of bio-inspired photoactive nanodevices

Capillary coating as an important factor in optimization of the off-line and on-line MEKC assays of the highly hydrophobic enzyme chlorophyllase

The choice between bare and coated capillaries is a key decision in the development and use of any methods based on capillary electrophoresis. In this work several permanently and dynamically coated capillaries were successfully implemented in a previously developed micellar electrokinetic chromatography (MEKC) assay of the plant membrane enzyme chlorophyllase. The results obtained demonstrate the rationale behind the use of capillary coating, which is crucial for successful optimization of both the off-line mode and the on-line/electrophoretically mediated microanalysis assay mode. The application of an amine permanently coated capillary (eCAP) is a simple way to significantly increase the repeatability of migration times and peak areas, and to ensure a strong electroosmotic flow that considerably decreases the overall analysis time. A dynamic coating (CEofix) allows one to apply an on-line incubation to control the reaction progress inside the capillary, and to increase the signal-to-noise ratio and peak efficiency. The dynamic coating is possible with use of both the normally applied uncoated silica capillary and the precoated amine capillary, which ensures more repeatable migration times. The strong points of the uncoated silica capillary are its attractive price and wide range of pH that can be applied. The characteristics presented may simplify the choice of capillary modification, especially in the case of hydrophobic analytes, MEKC-based separations, and other enzymatic assays. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-016-0097-5) contains supplementary material, which is available to authorized users

Reconstitution Approach to Tune Spectral Features of Light Harvesting Complexes for Improved Light Absorption and Energy Transfer

AbstractLight harvesting complexes developed by living organisms render themselves as an excellent system for understanding basic physical and chemical processes behind the conversion of sunlight energy. Although light harvesting complexes are pretty robust, biochemical reconstitution and genetic modifications have proven the flexibility to tailor their absorption spectra and energy transfer. Importantly, the refolding of the protein and the exchanging of the pigment in micellar media results in very similar pigment arrangement within the native complexes. Here, we show reconstitution approaches with different pigments that have been carried out in PCP, LHCII, and LHI complexes. Monitoring on the spectral changes and energy transfer has also been described

Optimization of Western blotting analysis for the isolation and detection of membrane xenobiotic transporter ABCG2

All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP-binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification. The aim of this study was to optimize the methods for detection and isolation of BCRP-enriched fractions obtained from animal tissue samples. In this report we describe an optimization of isolation of a BCRP-enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools

Lessons from chlorophylls : modifications of porphyrinoids towards optimized solar energy conversion

Practical applications of photosynthesis-inspired processes depend on a thorough understanding of the structures and physiochemical features of pigment molecules such as chlorophylls and bacteriochlorophylls. Consequently, the major structural features of these pigments have been systematically examined as to how they influence the S_{1} state energy, lifetimes, quantum yields, and pigment photostability. In particular, the effects of the macrocyclic π-electron system, central metal ion (CMI), peripheral substituents, and pigment aggregation, on these critical parameters are discussed. The results obtained confirm that the π-electron system of the chromophore has the greatest influence on the light energy conversion capacity of porphyrinoids. Its modifications lead to changes in molecular symmetry, which determine the energy levels of frontier orbitals and hence affect the S_{1} state properties. In the case of bacteriochlorophylls aggregation can also strongly decrease the S_{1} energy. The CMI may be considered as another influential structural feature which only moderately influences the ground-state properties of bacteriochlorophylls but strongly affects the singlet excited-state. An introduction of CMIs heavier than Mg^{2+} significantly improves pigments' photostabilities, however, at the expense of S_{1} state lifetime. Modifications of the peripheral substituents may also influence the S1 energy, and pigments’ redox potentials, which in turn influence their photostability

Pulmonary metastases of the A549-derived lung adenocarcinoma tumors growing in nude mice : a multiple case study

Lung adenocarcinoma is a leading human malignancy with fatal prognosis. Ninety percent of the deaths, however, are caused by metastases. The model of subcutaneous tumor xenograft in nude mice was adopted to study the growth of control and photodynamically treated tumors derived from the human A549 lung adenocarcinoma cell line. As a side-result of the primary studies, observations on the metastasis of these tumors to the murine lungs were collected, and reported in the present paper. The metastasizing primary tumors were drained by a prominent number of lymphatic vessels. The metastatic tissue revealed the morphology of well-differentiated or trans-differentiated adenocarcinoma. Further histological and histochemical analyses demonstrated the presence of golden-brown granules in the metastatic tissue, similar to these found in the tumor tissue. In contrast to the primary tumors, the electron paramagnetic resonance spectroscopy revealed no nitric oxide - hemoglobin complexes (a source of intense paramagnetic signals), in the metastases. No metastases were found in other murine organs; however, white infarctions were identified in a single liver. Taken together, the A549-derived tumors growing subcutaneously in nude mice can metastasize and grow on site in the pulmonary tissue. Thus, they can represent an alternative for the model of induced metastatic nodule formation, following intravenous administration of the cancerous cells