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Polyurea microcapsules containing glucose oxidase were prepared and their thermodurability was examined. Microencapsulated glucose oxidase was found to be more stable to heat than the enzyme in free solution. This stability was enhanced with an increase in the amount of enzyme entrapped in the microcapsules.
In the presence of the Cu(I)-chelating agent neocuproine (2,10-dimethyl-1,9-phenanthroline) hydrogen peroxide acts as a reductant of Cu(II). The reaction does not proceed in the absence of neocuproine and the addition of EDTA to the reaction mixture prior to addition of Cu(II) also inhibits the reduction. Colour development can be arrested and stabilized by addition of EDTA. The reaction can be used to estimate hydrogen peroxide concentrations in the range 0.68-6.8 micrograms/ml and glucose concentrations in the range 3.6-36 micrograms/ml (20-200 microM). Horseradish peroxidase is not required for the peroxide assay but glucose oxidase must be used for glucose estimations. Thermostable cellulase activity has been estimated at 60 degrees C against cellobiose, carboxymethylcellulose and cellulose substrates by estimation of the glucose released from the substrates.
5-Ethylphenazine-glucose-dehydrogenase-NAD+ conjugate (EP(+)-GlcDH-NAD+) was prepared by linking both poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to glucose dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.8, and that of the NAD+ moieties was 1.2. This conjugate is a semisynthetic enzyme having glucose oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following values of the kinetic constants: for the system with oxygen, the turnover number per subunit is 0.40 s-1 and Km for oxygen is 1.57 mM; and for the system with MTT, the turnover number is 0.11 s-1 and Km for MTT is 0.072 mM. The catalytic cycle of the semisynthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety. The apparent intramolecular rate constants of these steps were estimated, and the values are as follows: 0.39 s-1 for the reductions of the NAD+ moiety, 2.2 s-1 and 0.12 s-1 for the oxidation of the NADH moiety in the systems with oxygen and with MTT, respectively, and 3.2 s-1 and 0.18 s-1 for the reduction of the ethylphenazine moiety in the systems with oxygen and with MTT, respectively. On the bases of these results, the following three rate-acceleration mechanisms of the semisynthetic glucose oxidase are discussed: high effective concentration, intramolecular coupling of successive catalytic reactions, and multiple connection between the two kinds of the catalytic sites.
Previous studies have shown that several mixed-function oxidation (MFO) systems are capable of catalyzing the inactivation of glutamine synthetase (GS) [R.L. Levine, C. N. Oliver, R. M. Fulks, and E. R. Stadtman (1978) Proc. Natl. Acad. Sci. USA 78, 2120-2124] and a number of the other enzymes [L. Fucci, C. N. Oliver, M. J. Coon, and E. R. Stadtman (1983) Proc. Natl. Acad. Sci. USA 80, 1521-1525]. It has now been found that in the presence of Fe(III), O2, and an appropriate electron donor (hypoxanthine or NADPH, respectively) glutamine synthetase is also inactivated by either milk xanthine oxidase or Clostridial nicotinate hydroxylase. Inactivation of glutamine synthetase by either of these flavoproteins is greatly stimulated by the presence of electron carrier proteins possessing nonheme-iron-sulfur (NHIS) clusters (i.e., ferredoxin or putidaredoxin) or by the presence of menadione. The inactivation reactions are partially inhibited by free radical scavengers, superoxide dismutase, (SOD), histidine, mannitol, dimethyl sulfoxide, and dimethylthiourea, and are inhibited completely by either Mn(II), EDTA, or catalase. The sensitivity to SOD inhibition is greatly suppressed when the xanthine oxidase system is supplemented with either ferredoxin or redoxin. In the presence of the latter NHIS-proteins (and only when they are present), MFO systems, comprised of either horseradish peroxidase and H2O2 or glucose oxidase, O2, and glucose, can also catalyze the inactivation of GS. The ability of ferredoxin and putidaredoxin to promote oxidation modification of GS by any one of these MFO systems suggests that proteins with NHIS centers may mediate the generation (or stabilization) of highly reactive radical intermediates.
Glucose oxidase has been immobilized onto a thin platinum strip, by co-crosslinking with bovine serum albumin and glutaraldehyde. The retention of redox characteristics of glucose oxidase has been verified by cyclic voltammetry. The activity of the immobilized enzyme reduces to a quarter of its value when the enzyme is in solution but improves when coimmobilized with 1 M urea. The potentiometric response builds up and remains stable after 100 s. It is sensitive to the thickness of the immobilizing matrix. pH and temperature. An improvement in the performance of the electrode has been achieved by co-immobilizing 2 M urea and metal ions such as Mg2+ and Mn2+. The presence of Cu has been proved to be detrimental. The electrode has been calibrated in the 0.1-5.0 mM glucose concentration range. It gives a stable response for more than 50 independent assays and can be stored for 60 days without significant loss of function.
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Measurements of glucose in samples exhibiting high variability in oxygen content present an important problem to solve for pO2 detection methods. Different solutions exist, all based on the stabilization of the pO2 sample before or during the measurement. The electrode described in this paper itself contains enough O2 to compensate for the variability of the sample oxygen content. The enzyme is cross-linked with gelatin using the bifunctional agent, glutaraldehyde. Amperometric enzyme electrodes have been constructed using these membranes. Measurements have been done by collecting the derivative in time of the signal given by the electrode for blood or plasma samples.
BACKGROUND: The degradation of the glucose oxidase (GOD) enzyme, commonly used in the construction of glucose sensors has been of concern for scientists for decades. Many researchers have found that GOD deactivates over time, mostly due to H2O2 oxidation. This decay can lead to the eventual failure of the sensor. However, these findings are controversial, because other researchers did not find this degradation. METHODS: The goal of this study was twofold. The first goal was to evaluate the in vitro and in vivo stability of two commercially available GOD enzymes and the second goal was to evaluate Nafion as a protective coating of GOD. Crosslinked GOD samples were sandwiched between two 10-microm pore polycarbonate membranes (Nafion coated or uncoated) and placed in custom designed Lexan chambers. Chambers were then exposed to a total of five different environments: Dulbecco's Modified Eagle Medium (DMEM) or phosphate buffered saline (PBS) with and without a 5.6-mM glucose concentration, as well as the subcutaneous in vivo environment of 12 rats. After a period of up to 4 weeks, chambers were retrieved, opened, and tested for enzyme activity using a three-electrode system. RESULTS: Enzyme activity showed only a slight decrease when exposed to DMEM and PBS without glucose. A more dramatic decrease in activity was observed in enzymes exposed to PBS and DMEM with 5.6 mM glucose. The in vivo environment also caused a significant decrease in enzyme activity, but the decrease was lower than for the in vitro environment with glucose conditions. CONCLUSION: The presence of glucose in vitro and in vivo led to the production of H2O2, suggesting this to be the main agent responsible for enzyme degradation. The use of a Nafion coating did not provide any additional protection.
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Conjugates of antibody with glucose oxidase obtained via the carbohydrate moiety of the enzyme via oxidation with periodate are suggested as a tool for selective killing of the target cell. The conjugate was separated from uncoupled enzyme by repeated precipitation with ammonium sulfate (42% saturation). Purity of the conjugate was estimated by gel filtration on Toya Pearl TP-65. The glucose oxidase conjugated with rabbit antibody against mouse IgG bound specifically to plastic-adsorbed mouse immunoglobulins and to cultured human endothelial cells pretreated with mouse anti-endothelial antiserum. Glucose oxidase targeted to the cells generates hydrogen peroxide in the presence of glucose. This hydrogen peroxide killed the endothelial cells even in the absence of a halide-peroxidase system and in the presence of catalase. Features of the conjugate (specificity, effective cytotoxicity, high stability) make it suitable for prospective in vivo application and for immunoselective segregation of heterogeneous cell populations.
Previous work has shown that glucose oxidase can be immobilized on platinum to give an electrode that responds potentiometrically to glucose over the clinically useful range of about 10-250 mg glucose/100 mL. The present studies were carried out with electrochemically pretreated platinum and with gold or porous graphite substituted for the platinum support. The presence of the enzyme gave a significantly enhanced potentiometric response over that obtained with the bare support for both the pretreated platinum and the porous graphite, but not with gold. However, with platinum the potentiometric response became more negative with increasing glucose concentration. With porous graphite, the potential changed in the positive direction as the glucose concentration was increased. Hysteresis was demonstrated for the platinum-enzyme electrode. Mass transfer measurements with a rotating ring-disc electrode (RRDE) showed measurable diffusional resistances to the transport of a model electroactive compound (potassium ferrocyanide) through a matrix of immobilized enzyme attached to the disc of the RRDE. These results are part of a larger study to define the source of the potentiometric response by examining the roles of the support and the mass transfer resistances through the immobilized enzyme matrix.
The thermostability of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) at 60 degrees C has been studied as a function of its concentration in various media (pure water and pure deuterium oxide). In deuterium oxide, glucose oxidase is more stable than in water, and two kinds of stabilizing effect have been observed: the medium-organization effect and the enzyme-concentration effect. This effect has been related to the glucose oxidase subunit structure. This enzyme contains four forms of subunit: monomer, dimer, trimer, and tetramer, which are all composed of the identical monomer. The monomers of glucose oxidase subunits are linked by the non-covalent bond. Only dimer and trimer possess the enzymatic activity. During glucose oxidase denaturing, monomers assemble into dimer, trimer, or tetramer. This redistribution behavior depends on the enzyme concentration and the nature of the medium.