DFT-based prediction of reactivity of short-chain alcohol dehydrogenase.

The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, (S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2' ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.

Temperature-dependent bifurcation of cooperative interactions in pure and enriched in ß-carotene DPPC liposomes.

We examined the influence of temperature on lipid intermolecular interactions and the organization of bilayers within multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes. We also investigated the effect of 0.5 mol% ß-carotene, a non-polar carotenoid, on the adhesive properties of these liposomes. Atomic force microscopy (AFM) and differential scanning calorimetry (DSC) were used to correlate the changes in the physical properties of the liposomal systems with their thermotropic behaviour. Using DSC we detected two transitions in pure DPPC vesicles and in those containing 0.5 mol% ß-carotene. In both systems the pretransition occurred at 34.5(1)°C and the main phase transition at 41.4 °C during heating. Upon cooling, the temperatures of the pretransition and the main transition decreased by about 6 °C and 1 °C, respectively. Changes in enthalpy and entropy were also similar in the two investigated systems. Data obtained in parallel AFM force experiments show that the adhesive forces between the liposomal systems and AFM probe strongly depend on the loading rate. Moreover, their characteristic monotonic changes and discontinuities are sensitive to temperature. In the range of temperatures from 27 °C to 31 °C, i.e. below the temperature of phase transition from gel to ripple phase, the adhesive forces measured in a water environment are about an order of magnitude higher in the presence of ß-carotene than in pure DPPC liposomes. The observed variable dependence of adhesion on the loading rate suggests that there are changes in the long- and short-range interactions between lipids, and that these may be related to the occurrence of some clustering effects. In addition, the simultaneous existence of different subphases was found in the gel phase of DPPC liposomes. The presence of ß-carotene at a level of 0.5 mol% stimulates the structural reorganization of DPPC multilamellar vesicles and enhances the bifurcation phenomenon detected in these systems.

An in vitro study on the antioxidant capacity of usnic acid on human erythrocytes and molecular models of its membrane.

Usnic acid (UA) has been associated with chronic diseases through its antioxidant action. Its main target is the cell membrane; however, its effect on that of human erythrocytes has been scarcely investigated. To gain insight into the molecular mechanisms of the interaction between UA and cell membranes human erythrocytes and molecular models of its membrane have been utilized. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Results by X-ray diffraction showed that UA produced structural perturbations on DMPC and DMPE bilayers. DSC studies have indicated that thermotropic behavior of DMPE was most strongly distorted by UA than DMPC, whereas the latter is mainly affected on the pretransition. Scanning electron (SEM) and defocusing microscopy (DM) showed that UA induced alterations to erythrocytes from the normal discoid shape to echinocytes. These results imply that UA molecules were located in the outer monolayer of the erythrocyte membrane. Results of its antioxidant properties showed that UA neutralized the oxidative capacity of HClO on DMPC and DMPE bilayers; SEM, DM and hemolysis assays demonstrated the protective effect of UA against the deleterious oxidant effects of HClO upon human erythrocytes.

Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate.

Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50µM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25-1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.

Influence of the redox state of ubiquinones and plastoquinones on the order of lipid bilayers studied by fluorescence anisotropy of diphenylhexatriene and trimethylammonium diphenylhexatriene.

The measurements of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) fluorescence anisotropy in egg yolk lecithin (EYL) and of DPH anisotropy in dipalmitoylphosphatidylcholine (DPPC) liposomes containing different concentrations of oxidized and reduced ubiquinone (UQ) and plastoquinone (PQ) homologues have been performed. All the oxidized UQ homologues strongly induced ordering of EYL membrane structure, whereas in DPPC liposomes, above the phase transition temperature, the most pronounced effect showed UQ-4. PQ-2 and PQ-9 were less effective than the corresponding ubiquinones in this respect. The reduced forms of UQ and PQ homologues increased the order of membrane lipids to a smaller extent than the corresponding quinones both in the interior of the membrane and closer to its surface. Nevertheless, the investigated prenylquinols showed stronger increase in the membrane order than alpha-tocopherol or alpha-tocopherol acetate, which could be connected with binding of prenylquinol head groups to phospholipid molecules by hydrogen bonds. The strong ordering influence of ubiquinones on the membrane structure was attributed to methoxyl groups of the UQ quinone rings.

Location of ubiquinone homologues in liposome membranes studied by fluorescence anisotropy of diphenyl-hexatriene and trimethylammonium-diphenyl-hexatriene.

The measurements of diphenyl-hexatriene (DPH) and trimethylammonium-diphenyl-hexatriene (TMA-DPH) fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) and egg yolk lecithin (EYL) liposomes containing different concentrations of various ubiquinone (UQ) homologues have been performed. UQ-4 induced the highest DPH anisotropy increase in DPPC liposomes, whereas for higher UQ homologues the anisotropy was lowered with the increase of UQ side-chain length. These differences were less pronounced in EYL liposomes. It was concluded that at a higher content in the membranes (3-4 mol%), the short-chain ubiquinones are arranged parallel to lipid fatty acid chains, whereas long-chain homologues are progressively removed from the lipid acyl chains into the midplane region of the membrane. At the lower (1-2 mol%) concentrations, long-chain quinones seem to be evenly distributed within the membrane, especially in EYL membranes. UQ-10 in EYL liposomes perturbed TMA-DPH to a similar extend as the short-chain ubiquinones indicating that UQ-10 penetrates the interface regions of the membrane where its redox reactions occur. The localization and physical state of UQ-10 in native membranes is discussed.