RESUMEN

Iron porphyrins play several important roles in present-day living systems and probably already existed in very early life forms. Hemin (= ferric protoporphyrin IX = ferric heme b), for example, is the prosthetic group at the active site of heme peroxidases, catalyzing the oxidation of a number of different types of reducing substrates after hemin is first oxidized by hydrogen peroxide as the oxidizing substrate of the enzyme. The active site of heme peroxidases consists of a hydrophobic pocket in which hemin is embedded noncovalently and kept in place through coordination of the iron atom to a proximal histidine side chain of the protein. It is this partially hydrophobic local environment of the enzyme which determines the efficiency with which the sequential reactions of the oxidizing and reducing substrates proceed at the active site. Free hemin, which has been separated from the protein moiety of heme peroxidases, is known to aggregate in an aqueous solution and exhibits low catalytic activity. Based on previous reports on the use of surfactant micelles to solubilize free hemin in a nonaggregated state, the peroxidase-like activity of hemin in the presence of sodium dodecyl sulfate (SDS) at concentrations below and above the critical concentration for SDS micelle formation (critical micellization concentration (cmc)) was systematically investigated. In most experiments, 3,3',5,5'-tetramethylbenzidine (TMB) was applied as a reducing substrate at pH = 7.2. The presence of SDS clearly had a positive effect on the reaction in terms of initial reaction rate and reaction yield, even at concentrations below the cmc. The highest activity correlated with the cmc value, as demonstrated for reactions at three different HEPES concentrations. The 4-(2-hydroxyethyl)-1-piperazineethanesulfonate salt (HEPES) served as a pH buffer substance and also had an accelerating effect on the reaction. At the cmc, the addition of l-histidine (l-His) resulted in a further concentration-dependent increase in the peroxidase-like activity of hemin until a maximal effect was reached at an optimal l-His concentration, probably corresponding to an ideal mono-l-His ligation to hemin. Some of the results obtained can be understood on the basis of molecular dynamics simulations, which indicated the existence of intermolecular interactions between hemin and HEPES and between hemin and SDS. Preliminary experiments with SDS/dodecanol vesicles at pH = 7.2 showed that in the presence of the vesicles, hemin exhibited similar peroxidase-like activity as in the case of SDS micelles. This supports the hypothesis that micelle- or vesicle-associated ferric or ferrous iron porphyrins may have played a role as primitive catalysts in membranous prebiotic compartment systems before cellular life emerged.

RESUMEN

The Trametes versicolor laccase (TvL)-catalysed oligomerisation of the aniline dimer p-aminodiphenylamine (PADPA) was investigated in an aqueous medium of pH = 3.5, containing 80-100 nm-sized anionic vesicles formed from AOT, the sodium salt of bis(2-ethylhexyl)sulfosuccinic acid. If run under optimal conditions, the reaction yields oligomeric products which resemble the emeraldine salt form of polyaniline (PANI-ES) in its polaron state, known to be the only oxidation state of linear PANI which is electrically conductive. The vesicles serve as "templates" for obtaining products with the desired PANI-ES-like features. For this complex, heterogeneous, vesicle-assisted, and enzyme-mediated reaction, in which dissolved dioxygen also takes part as a re-oxidant for TvL, small changes in the composition of the reaction mixture can have significant effects. Initial conditions may not only affect the kinetics of the reaction, but also the outcome, i.e., the product distribution once the reaction reaches its equilibrium state. While a change in the reaction temperature from T ≈ 25 to 5 °C mainly influenced the rate of reaction, increase in enzyme concentration and the presence of millimolar concentrations of chloride ions were found to have significant undesired effects on the outcome of the reaction. Chloride ions, which may originate from the preparation of the pH = 3.5 solution, inhibit TvL, such that higher TvL concentrations are required than without chloride to yield the same product distribution for the same reaction runtime as in the absence of chloride. With TvL concentrations much higher than the elaborated value, the products obtained clearly were different and over-oxidised. Thus, a change in the activity of the enzyme was found to have influence not only on kinetics but also led to a change in the final product distribution, molecular structure and electrical properties, which was a surprising find. The complementary analytical methods which we used in this work were in situ UV/vis/NIR, EPR, and Raman spectroscopy measurements, in combination with a detailed ex situ HPLC analysis and molecular dynamics simulations. With the results obtained, we would like to recall the often neglected or ignored fact that it is important to describe and pay attention to the experimental details, since this matters for being able to perform experiments in a reproducible way.

RESUMEN

Cholesterol (Chol-OH) and its conjugates are powerful molecules for engineering the physicochemical and magnetic properties of phospholipid bilayers in bicelles. Introduction of aminocholesterol (3ß-amino-5-cholestene, Chol-NH2) in bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the thulium-ion-chelating phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA/Tm3+) results in unprecedented high magnetic alignments by selectively tuning the magnetic susceptibility Δχ of the bilayer. However, little is known on the underlying mechanisms behind the magnetic response and, more generally, on the physicochemical forces governing a Chol-NH2 doped DMPC bilayer. We tackled this shortcoming with a multiscale bottom-up comparative investigation of Chol-OH and Chol-NH2 mixed with DMPC. First, simplified monolayer models on a Langmuir trough were employed to compare the two steroid molecules at various contents in DMPC. In a second step, a molecular dynamics (MD) simulation allowed for a more representative model of the bicelle bilayer while monitoring the amphiphiles and their interactions on the molecular level. In a final step, we moved away from the models and investigated the effect of temperature on the structure and magnetic alignment of Chol-NH2 doped bicelles by SANS. The DMPC/steroid monolayer studies showed that Chol-OH induces a larger condensation effect than Chol-NH2 at steroid contents of 16 and 20 mol %. However, this tendency was inversed at steroid contents of 10, 30, and 40 mol %. Although the MD simulation with 16 mol % steroid revealed that both compounds induce a liquid-ordered state in DMPC, the bilayer containing Chol-NH2 was much less ordered than the analogous system containing Chol-OH. Chol-NH2 underwent significantly more hydrogen bonding interactions with neighboring DMPC lipids than Chol-OH. It seems that, by altering the dynamics of the hydrophilic environment of the bicelle, Chol-NH2 changes the crystal field and angle of the phospholipid-lanthanide DMPE-DTPA/Tm3+ complex. These parameters largely determine the magnetic susceptibility Δχ of the complex, explaining the SANS results, which show significant differences in magnetic alignment of the steroid doped bicelles. Highly magnetically alignable DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles were achieved up to temperatures of 35 °C before a thermoreversible rearrangement into nonalignable vesicles occurred. The results confirm the potential of Chol-NH2 doped bicelles to act as building blocks for the development of the magnetically responsive soft materials of tomorrow.

Asunto(s)

RESUMEN

The oxidation of the aniline dimer, p-aminodiphenylamine (PADPA), with Trametes versicolor laccase and O2 in an aqueous solution of pH 3.5 is controlled by negatively charged AOT (sodium bis(2-ethylhexyl) sulfosuccinate) vesicles. With vesicles, a product resembling polyaniline in its emeraldine salt form (PANI-ES) is obtained, in contrast to the reaction without vesicles where no such product is formed. To understand this observation, the product distribution and structures from the reaction with and without vesicles were determined by using partially selectively deuterated PADPA as a starting material and analyzing the products with HPLC-MS. We found that in the presence of vesicles the main product is obtained in about 50% yield, which is the N-C-para-coupled PADPA dimer that has spectroscopic properties of PANI-ES, as determined by time-dependent density functional theory (TD-DFT) calculations. A secondary reaction route leads to longer PADPA oligomers that must contain a phenazine core. Without vesicles, PADPA and its products undergo partial hydrolysis, but in the presence of vesicles, hydrolysis does not occur. Because molecular dynamics (MD) simulations show that the main intermediate oxidation product is embedded within the vesicle membrane, where the water content is very low, we propose that the microenvironment of the vesicle membrane protects the oxidation products from unwanted hydrolysis.

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RESUMEN

The performance of the GROMOS96 parameter set 45A3 developed for aliphatic alkanes is tested on a bilayer of dipalmitoylphosphatidylcholine (DPPC) in water in the liquid-crystalline L(alpha) phase. Variants of the force-field parameter set as well as different sets of simulation conditions or simulation parameter sets are evaluated. In the case of the force-field parameters, the van der Waals constants for the non-bonded interaction of the ester carbonyl carbon and the partial charges and charge group definition of the phosphatidylcholine head group are examined. On the methodological side, different cut-off distances for the non-bonded interactions, use of a reaction-field force due to long-range electrostatic interactions, the frequency of removal of the centre of mass motion and the strength of the coupling of the pressure of the system to the pressure bath are tested. The area per lipid, as a measure of structure, the order parameters of the chain carbons, as a measure of membrane fluidity, and the translational diffusion of the lipids in the plane of the bilayer are calculated and compared with experimental values. An optimal set of simulation parameters for which the GROMOS96 parameter set 45A3 yields a head group area, chain order parameters and a lateral diffusion coefficient in accordance with the experimental data is listed.

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