Chitosan effects on monolayers of zwitterionic, anionic and a natural lipid extract from E. coli at physiological pH.

Langmuir monolayers are used to simulate the biological membrane environment, acting as a mimetic system of the outer or the inner membrane leaflet. Herein, we analyze the interaction of membrane models with a partially N-acetylated chitosan (Ch35%) possessing a quasi-ideal random pattern of acetylation, full water solubility up to pH ≈ 8.5 and unusually high weight average molecular weight. Lipid monolayers containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethalonamine (DPPE), dipalmitoyl phosphatidyl glycerol (DPPG) or E. coli total lipid extract were spread onto subphases buffered at pH 4.5 or 7.4. The incorporation of Ch35% chitosan caused monolayer expansion and a general trend of decreasing monolayer rigidity with Ch35% concentration. Due to its relatively high content of N-acetylglucosamine (GlcNAc) units, Ch35% interactions with negatively charged monolayers and with E. coli extract were weaker than those involving zwitterionic monolayers or lipid rafts. While the smaller interaction with negatively charged lipids was unexpected, this finding can be attributed to the degree of acetylation (35%) which imparts a small number of charged groups for Ch35% to interact. Chitosan properties are therefore determinant for interactions with model cell membranes, which explains the variability in chitosan bactericide activity in the literature. This is the first study on the effects from chitosans on realistic models of bacterial membranes under physiological pH.

Cholesterol modulates the interaction between paclitaxel and Langmuir monolayers simulating cell membranes.

The composition of Langmuir monolayers used as cell membrane models is an essential factor for the interaction with biologically-relevant molecules, including pharmaceutical drugs. In this paper, we report the modulation of effects from the antineoplastic drug paclitaxel by the relative concentration of cholesterol in the Langmuir monolayers of ternary mixtures of dipalmitoylphosphatidylcholine, sphingomyelin, and cholesterol. Since the dependence on cholesterol concentration for these monolayers simulating lipid rafts is non-monotonic, we analyzed the surface pressure and compressibility modulus data with the multidimensional projection technique referred to as interactive document mapping (IDMAP). The maximum expansion induced by paclitaxel in surface pressure isotherms was observed for 27% cholesterol, while the compressibility modulus decreased most strongly for the monolayer with 48% cholesterol. Therefore, the physiological action of paclitaxel may vary depending on whether it is associated with penetration in the membrane or with changes in the membrane elasticity.

Nanosized non-proteinaceous complexes III and IV mimicking electron transfer of mitochondrial respiratory chain.

Synthetic biology pursues the understanding of biological processes and their possible mimicry with artificial bioinspired materials. A number of materials have already been used to mimic the active site of simple redox proteins, including nanosized iron oxides due to their redox properties. However, the mimicry of membrane redox protein complexes is still a challenge. Herein, magnetic iron oxide nanoparticles (NPs), incorporated as non-proteinaceous complexes III and IV in a mitochondrial model membrane, catalyze electron transfer (ET) similarly to the natural complexes towards cytochrome c. The associated molecular mechanism is experimentally proven in solution and in a Langmuir-Blodgett film. A direct and entropy-driven ET, with rate constant of 2.63 ± 0.05Lmol-1 at 25 °C, occurs between the iron sites of the NPs and the cytochrome c heme group, not affecting the protein secondary and tertiary structures. This process requires an activation energy of 40.2 ± 1.5 kJ mol-1 resulting in an overall Gibbs free energy of -55.3 kJ mol-1. Furthermore, the protein-NP system is governed by electrostatic and non-polar forces that contribute to an associative mechanism in the transition state. Finally, the incorporated NPs in a model membrane were able to catalyze ET, such as the natural complexes in respiratory chain. This work presents an experimental approach demonstrating that inorganic nanostructured systems may behave as embedded proteins in the eukaryotic cells membrane, opening the way for more sophisticated and robust mimicry of membrane protein complexes.

Enhanced chitosan effects on cell membrane models made with lipid raft monolayers.

Langmuir monolayers have been used as cell membrane models, where lipid composition is normally varied to mimic distinct types of membranes. For eukaryotic membranes, for instance, rather than using only zwitterionic phospholipids there is now a trend to employ mixtures to simulate the lipid rafts known to be relevant for various cellular processes. In this study, we demonstrate that effects from chitosans on Langmuir monolayers are considerably higher if lipid raft compositions (ternary mixtures of dipalmitoyl phosphatidyl choline (DPPC), sphingomyelin (SM) and cholesterol) are used. Significantly, measurable effects on the surface pressure isotherms start at 10-6 mg mL-1 for chitosans in lipid rafts, to be compared with 10-2 mg mL-1 for neat dipalmitoyl phosphatidylcholine (DPPC). This applies to both a commercial chitosan and chitosans soluble at physiological pH. Incorporation of these chitosans in the raft monolayers was confirmed in polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) experiments, where both the tail groups and headgroups were found to interact with chitosan. Since the effects on membrane models may be observed at such small concentrations for chitosans and probably other molecules, some studies may have to be revisited where neat phospholipids should be replaced by lipid raft compositions.

Advances in enzyme bioelectrochemistry.

Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans) where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

Ethanol generation, oxidation and energy production in a cooperative bioelectrochemical system.

Integrating in situ biofuel production and energy conversion into a single system ensures the production of more robust networks as well as more renewable technologies. For this purpose, identifying and developing new biocatalysts is crucial. Herein, is reported a bioelectrochemical system consisting of alcohol dehydrogenase (ADH) and Saccharomyces cerevisiae, wherein both function cooperatively for ethanol production and its bioelectrochemical oxidation. Here, it is shown that it is possible to produce ethanol and use it as a biofuel in a tandem manner. The strategy is to employ flexible carbon fibres (FCF) electrode that could adsorb both the enzyme and the yeast cells. Glucose is used as a substrate for the yeast for the production of ethanol, while the enzyme is used to catalyse the oxidation of ethanol to acetaldehyde. Regarding the generation of reliable electricity based on electrochemical systems, the biosystem proposed in this study operates at a low temperature and ethanol production is proportional to the generated current. With further optimisation of electrode design, we envision the use of the cooperative biofuel cell for energy conversion and management of organic compounds.

Advances in enzyme bioelectrochemistry

ABSTRACT Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans) where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

Evidence of short-range electron transfer of a redox enzyme on graphene oxide electrodes.

Direct electron transfer (DET) between redox enzymes and electrode surfaces is of growing interest and an important strategy in the development of biofuel cells and biosensors. Among the nanomaterials utilized at electrode/enzyme interfaces to enhance the electronic communication, graphene oxide (GO) has been identified as a highly promising candidate. It is postulated that GO layers decrease the distance between the flavin cofactor (FAD/FADH2) of the glucose oxidase enzyme (GOx) and the electrode surface, though experimental evidence concerning the distance dependence of the rate constant for heterogeneous electron-transfer (k(het)) has not yet been observed. In this work, we report the experimentally observed DET of the GOx enzyme adsorbed on flexible carbon fiber (FCF) electrodes modified with GO (FCF-GO), where the k(het) between GO and electroactive GOx has been measured at a structurally well-defined interface. The curves obtained from the Marcus theory were used to obtain k(het), by using the model proposed by Chidsey. In agreement with experimental data, this model proved to be useful to systematically probe the dependence of electron transfer rates on distance, in order to provide an empirical basis to understand the origin of interfacial DET between GO and GOx. We also demonstrate that the presence of GO at the enzyme/electrode interface diminishes the activation energy by decreasing the distance between the electrode surface and FAD/FADH2.

Molecular interactions and structure of a supramolecular arrangement of glucose oxidase and palladium nanoparticles.

This paper presents studies about the molecular interactions and redox processes involved in the formation of palladium nanoparticles associated to glucose oxidase (GOx-PdNPs) in a supramolecular arrangement. The synthesis occurs in two steps, the Pd reduction and the formation of the 80 nm sized supramolecular aggregates containing multiples units of GOx associated to 3.5 nm sized PdNPs. During synthesis, GOx molecules interact with Pd salt leading to metal ion and FAD reduction probably via the thiol group of the cysteine 521 residue. For the growing of PdNPs, formic acid was necessary as a co-adjuvant reducing agent. Besides the contribution for the redox processes, GOx is also necessary for the NP stability preventing the formation of precipitates resulted from uncontrolled growing of NPs Cyclic voltammetry of the GOx-PdNPs demonstrated electroactivity of the bionanocomposite immobilized on ITO (indium-tin oxide) electrode surface and also the NP is partially blocked due to strong interaction GOx and the surface of PdNPs. Vibrational spectroscopy (FTIR) showed that significant structural changes occurred in GOx after the association to PdNP. These mechanistics and structural studies can contribute for modulation of bionanocomposites properties.