Direct electron transfer and electrochemical study of hemoglobin immobilized in ZnO hollow spheres

ZnO hollow spheres were firstly prepared. A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping Hemoglobin (Hb) through the ZnO hollow spheres (ZHS) nanoparticles. The composition morphology and size were studied by transmission electron microscopy. The surface topography of the prepared films was imaged by atomic force microscope (AFM). Several techniques, including UV–vis absorption spectroscopy, cyclic voltammetry, chronoamperometry were employed to characterize the performance of the biosensor. The results indicated that the ZHS nanoparticles had enhanced the performance of the hydrogen peroxide sensors. The electrochemical parameters of Hb in the ZHS were calculated by the results of the electron-transfer coefficient (α) and the apparent heterogeneous electron-transfer rate constant K _s as 0.5 and 3.1 s⁻¹, respectively. The resulting biosensors showed a wide linear range from 2.1 × 10⁻⁶ to 5.18 × 10⁻³ M, with a low detection limit of 7.0 × 10⁻⁷ M (S/N = 3) under optimized experimental conditions. The results demonstrated that the ZHS matrix may improve the protein loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its unique morphology, high specific surface area, and biocompatibility. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability.     

Covalent immobilization of FAD and glucose oxidase on carbon electrodes

The effectiveness of attaching flavin adenine dinucleotide (FAD) via a C bridge to Teflon-bonded carbon black (CB), and the subsequent immobilization of glucose oxidase on the FAD-modified electrodes has been studied by cyclic voltammetry. When FAD alone is bound to the electrode, it undergoes reduction and oxidation at -0.62 and -0.5 V, respectively-values similar to those obtained with free FAD. Compared to the free enzyme, the reduction of FAD as part of the immobilized enzyme is 200 mV more cathodic, while the oxidation potential remains the same in both cases.     

Modification of hemoglobin by glutaric aldehyde and electrochemical properties of the conjugates

Electrochemical properties of macromolecules of modified haemoglobin obtained by polycondensation with glutaric aldehyde have been investigated by means of potentiometric titration, PAG-electrophoresis, ion-exchange chromatography, and (for evaluation of isoelectric point) distribution between two aqueous polymeric phases. Introduction of additional functional groups into the macromolecule is possible by using various agents blocking polycondensation, which makes it possible to change the resulting charge and the isoelectric point.     

The further preparation of inorganic cationic yeasts and some of their chief properties

1. A method is described for replacing the intracellular K(+) of the yeast cell by Rb(+), Cs(+), Li(+) or Ca(2+) ions. In the formation of a calcium yeast it is necessary to proceed first through a sodium yeast (Conway & Moore, 1954) as in the formation of a magnesium yeast (Conway & Beary, 1962). This concludes the series of such yeasts in which almost all the usual K(+) is replaced by another cation, and for which the effect on the properties of fermentation, oxygen uptake and of growth are described. 2. Previous work has shown that all these inorganic cations that can be accumulated in quantity at pH7.0 are taken up by the same carrier, that the uptake of Mg(2+) is almost completely inhibited by anoxia and cyanide (0.2mm) and that in the uptake of Mg(2+) ions a practically equivalent amount of H(+) ions is excreted. It is suggested that these facts amount to a definitive demonstration that the carrier is a cytochrome.     

Investigation of polypyrrole/glucose oxidase electrodes by ellipsometric, microgravimetric and electrochemical measurements

We describe the simultaneous application of two in-situ techniques for the study of the electrochemical growth of a conducting polymer film (polypyrrole) in the presence of an enzyme (glucose oxidase). The combination of optical (ellipsometric) and microgravimetric (QCMB) measurements employed in this study provides information on fundamental properties of the enzyme-containing film, including film thickness, mass and density. Our results show that incorporation of the enzyme results in changes in the apparent optical properties and in the apparent density of the electrochemically grown film which suggest mutual stabilization of the polypyrrole and the enzyme in the composite layer.     

Alizarin interaction with sickle hemoglobin: elucidation of their anti-sickling properties by multi-spectroscopic and molecular modeling techniques

Abstract

Polymerization of hemoglobin S is a major cause of morbidity and mortality in sickle cell disease, which leads to sickling and destruction of red blood cell. Alizarin, a bioactive compound from Rubia cordifolia, is reported to be blood purifier. This study investigates the potential of alizarin as an anti-sickling agent, showing a significant decrease in the rate of polymerization, therefore inhibiting the rate of sickling with increasing concentration. Interaction studies indicated that the fluorescence intensity of sickle hemoglobin (Hb S) decreases gradually with increasing alizarin concentration. This suggests the static quenching, where binding constant and the number of binding sites were deduced at different temperatures. The negative values of Gibbs energy change (ΔG⁰) strongly suggest that it is entropy-driven spontaneous and exothermic reaction. Negative enthalpy (ΔH⁰) and positive entropy (ΔS⁰) stipulated that hydrogen and hydrophobic bonding forces were interfering in a hydrophobic micro-environment of β6Val leading to Hb S polymerization inhibition. In circular dichroism (CD) spectra, Hb S in the presence of alizarin shows helical structural changes leading to destabilization of Hb S polymer. These findings were also supported by molecular docking simulation studies using DOCK6 and GROMACS. So, from these findings, we may conclude that alizarin interacts with Hb S through hydrogen bonding and leading to inhibition of Hb S polymerization. Consequently, alizarin may have potential use as an anti-sickle cell medication for sickle cell disorder.

Communicated by Ramaswamy H. Sarma     

Imaging of ATP membrane transport with dual micro-disk electrodes and scanning electrochemical microscopy

Extracellular adenosine-5'-triphosphate (ATP) is involved in a variety of relevant regulatory mechanisms at a cellular level and has therefore been focus of extensive research. One of the major challenges associated with measuring this key regulatory analyte is the ability to detect and localize extracellular ATP with sufficient spatial and temporal resolution in physiological environments. In this study, scanning electrochemical microscopy (SECM) utilizing an amperometric micro-biosensor based on co-immobilization of the enzymes glucose oxidase and hexokinase is applied for imaging ATP transport through a porous polycarbonate membrane under physiologically relevant conditions. The enzymatic biosensor operates on competitive consumption of the substrate glucose between the immobilized enzymes glucose oxidase and hexokinase involving ATP as a co-substrate. Quantitative determination of the ATP concentration is based on a linear correlation between the glucose consumption and the ATP level. Integration of the amperometric ATP micro-biosensor into a dual micro-disk electrode configuration is achieved by immobilizing the enzymes at one of the micro-disk electrodes while the second disk serves as an unmodified amperometric probe for controlled positioning of the micro-biosensor in close proximity to the sample surface enabling quantification of the obtained current signal.     

Direct voltammetric investigation of the electrochemical properties of human hemoglobin: relevance to physiological redox chemistry

Voltammetric measurements on solutions of human hemoglobin using gold electrodes modified with omega-hydroxyalkanethiols have yielded the first direct measure of the reorganization energy of the protein. The value obtained based on extrapolation of the experimentally measured currents, 0.76 eV, is independent of pH (i.e., over the physiologically relevant rage, pH 6.8-7.4) and is remarkably similar to values obtained for myoglobin. This result is perhaps surprising given the marked dependence of the measured reduction potential of hemoglobin on pH (i.e., the redox Bohr effect). Electron transfer rates from the electrode to hemoglobin were also measured. Using similarly measured heterogeneous electron-transfer rates for cytochrome b(5), it is possible to predict the magnitude of the homogeneous electron-transfer rate from cytochrome b(5) to methemoglobin using a formalism developed by Marcus. These predicted rates are in reasonable agreement with reported rates of this physiological reaction based on stopped-flow kinetics experiments. These results suggest that the intrinsic electroreactivity of these heme proteins is sufficient to account for physiologically observed rates. Residual differences between homogeneous phase kinetics and those predicted by heterogeneous phase reactions are suggested to be due to small reductions in the outer-sphere reorganization energy of both component proteins which arise due to solvent exclusion at the interface between the two proteins in complex.     

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant K_A = 7.0(± 0.8) × 10⁴ M^− 1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.     

Development of easy made low cost bindless monolithic electrodes from biomass with controlled properties to be used as electrochemical capacitors

The aim of the work now reported is the development of low cost electrodes in the monolithic shape without the need for a pos-production step with potential to be used in supercapacitors. The tested materials were activated carbon fibres prepared and activated carbons made from coffee endocarp. The main functional groups identified were quinone, lactone, Si-H, phenol, hydroxyl, carbonyl and ether for activated carbon samples and amine, amide, pyrone, lactone, carbonyl and hydroxyl for activated carbon fibres samples. The nanostructure of the materials is predominantly microporous but with a significant variety of porosity development with BET surface area and pore volume given by α(s) method range from 89 to 1050 m(2) g(-1) and 0.04 to 0.50 cm(3) g(-1), respectively. The electrochemical properties of the materials were investigated using classic cyclic voltammetry, chronopotentiometry and electrochemical impedance spectroscopy. The higher specific capacitance achieved was 176 F g(-1).