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Biomedical subjects

A E Cass

Publications and source records attributed to A E Cass.

At least 19 recordsLinked to original sources
PubMed1992

Partition coefficient of luciferase from photobacteria in PEG/salt two aqueous phase system.

The relationship between the logarithmic partition coefficient (K) of luciferase of photobacteria and PEG MW in the PEG/salt two aqueous phase system was shown to be a linear function. The hydrophilic PEG of lower MW facilitated the partition of luciferase into the top PEG-riching phase and gave a higher K value, while PEG of higher MW, its hydrophobic characteristic, made more enzyme partition into the bottom salt-riching phase and lower K value was obtained. In the PEG/trivalent salt system, such as phosphate and citrate, there was a turning-point on the linear relation between the log K and the PEG MW, but which never appeared if a divalent salt such as sulfate, succinate or tartrate was used in the system. When the system was composed of homogeneous PEG and ammonium sulfate, the K value was increased with the increment of the salt concentration, but after the salt concentration had reached at certain level, the K value was uninfluenced. When two kinds of PEG with different MW were used in this system a minimal K value appeared at certain concentration of ammonium sulfate, and the K value was raised when the salt concentration was either increased or decreased. Neither the proportion of the two kinds of PEG nor their total concentration used in the system showed any effect on the above patterns, although the K value may give some corresponding changes. (ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗
PubMed1990

Radical intermediates in veratryl alcohol oxidation by ligninase. NMR evidence.

Proton nuclear magnetic resonance (NMR) spectra of veratryl alcohol (3,4-dimethoxybenzyl alcohol) were obtained during its oxidation by ligninase. It was observed that a substantial increase in the linewidths of the resonances occurred only in the presence of both the enzyme and hydrogen peroxide. Quenching the reaction by the addition of alkali immediately restored the normal linewidths of the resonances. Furthermore, inversion-recovery experiments showed a decrease in the longitudinal relaxation time of the substrate when the enzyme was actively turning over. Changes in both these NMR parameters are consistent with the generation of radical intermediates during the ligninase-catalysed oxidation of veratryl alcohol.

Agaricales↗
PubMed1990

Cyanide detection using a substrate-regenerating, peroxidase-based biosensor.

An enzyme-based, dual working electrode system is described for the sensing of cyanide. Horseradish peroxidase (HRP) is incorporated as the sensing element. A continuous monitoring of oxidative activity by the enzyme results through the generation and regeneration of substrates at the electrode surfaces. Thus, HRP is oxidized by hydrogen peroxide generated from dissolved oxygen, at the primary electrode, and then reduced through the secondary electrode by mediated electron transfer using ferrocene as a carrier. Ferrocene regeneration at this electrode is proportional to the intrinsic activity of HRP. The dynamics of the system are investigated by using a rotating ring-disk electrode. The enzyme is immobilized to provide better control over its catalytic activity and to increase the lifetime of the biosensor. Cyanide inhibition of current can be modeled by reversible binding kinetics. Detection of cyanide is possible in submicromolar (ppb) concentrations, with a half maximal response at 2 microM. The response time for detection of introduced cyanide is within 1 s. The sensor can be operated between 5 and 40 degrees C, and cyanide inhibition is unaffected by pH changes between 5 and 8. The sensor is reproducible for cyanide determination and is stable for over 6 months.

Biosensing Techniques↗
PubMed1990

Inhibited enzyme electrodes. Part 1: Theoretical model.

A theoretical model is developed for an electrochemical sensor for toxic substances which works by measuring the inhibition of the enzyme activity. The enzyme is assumed to follow Michaelis-Menten kinetics and the diffusion kinetic equation describing the concentration profile of the enzyme's substrate in the electrolyte layer between the electrode and the membrane covering the electrode is solved. A complete set of analytical solutions is found which corresponds to a number of different rate limiting processes. The set of solutions is described in a case diagram. The use of cytochrome oxidase in particular is discussed.

Biosensing Techniques↗
PubMed1990

Inhibited enzyme electrodes. Part 2: The kinetics of the cytochrome oxidase system.

An inhibition enzyme electrode to measure toxic gases can be constructed using the respiratory enzyme cytochrome oxidase. The rate of enzyme turnover is followed by reducing cytochrome c on a gold electrode modified with the mediator bis(4-pyridyl) disulphate. The kinetics and mechanism of the system have been measured. The electrochemical kinetics for the oxidation of cytochrome c have been studied by rotating disc voltammetry and are shown to obey the Koutecky-Levich equation. The standard electrochemical rate constant is found to be 3 x 10(-3) cms-1. At ambient oxygen concentration the orders of the current with respect to the concentration of cytochrome oxidase, cytochrome c and oxygen are found to be 1/2, 1/2 and zero respectively. These orders are consistent with the rate limiting step being the turnover of the enzyme under saturated conditions in a thin reaction layer close to the electrode. At lower oxygen concentrations a good fit between the experimental results and a theoretical model further confirms the assignation of the mechanism. The rate constants describing the oxidation and reduction of the enzyme have been measured. The pH dependence of the current has been studied.

Biosensing Techniques↗
PubMed1990

Inhibited enzyme electrodes. Part 3. A sensor for low levels of H2S and HCN.

It is shown that an inhibited enzyme electrode, using cytochrome oxidase, will respond to H2S, HCN and azide ion. For all three inhibitors the kinetics of the inhibiton and recovery processes have been analysed using the theoretical model presented previously (Albery et al., 1990a). Rearrangement of the differential equation describing inhibition and the development of the necessary software has enabled us to obtain values of the concentration of inhibitor in a matter of seconds after exposure of the sensor. The sensor will measure concentrations of H2S down to 1 ppm in the gas phase and concentrations of HCN and azide ion down to 0.4 mumol dm-3 in the solution phase.

Azides↗
PubMed1990

Method for determining paracetamol in whole blood by chronoamperometry following enzymatic hydrolysis.

A method is proposed for the determination of paracetamol in whole undiluted blood, based on the enzymatic hydrolysis of the drug to p-aminophenol, which is then measured by chronoamperometry at a glassy carbon electrode. Hydrolysis of the paracetamol prior to electro-oxidation is shown to alleviate problems that arise from high background currents in the whole blood and so ensures a good linear correlation (r greater than 0.99) between the current and the paracetamol concentration. Recovery experiments and comparison with a reference method based on spectrophotometry suggest that the electrochemical assay only measures that proportion of paracetamol that is not bound to serum albumin.

Acetaminophen↗
PubMed1987

Amperometric enzyme electrodes.

Three different types of amperometric enzyme electrode are described. The first type uses a conducting organic-salt electrode to oxidize NADH. Results for sensors for ethanol and for bile acids are presented. In the second type of sensor, flavoenzymes are directly oxidized on the surface of the conducting organic-salt electrode. Results for five different enzymes are described. The mechanism of the enzyme oxidation is discussed and the reaction is shown to take place by heterogeneous redox catalysis and not by homogeneous mediation. The enzymes are strongly adsorbed on the electrode; microelectrodes for in vivo studies can be constructed without a membrane. Results for in vivo studies of changing glucose levels in the brain of a freely moving rat are presented. The third type of sensor is designed to measure low levels of toxic gases such as H2S and HCN. This is done by monitoring the inhibition by the toxic gas of the activity of the respiratory enzyme cytochrome oxidase.

Animals↗
PubMed1987

Biotransformation of aromatic compounds. Monitoring fluorinated analogues by NMR.

The results presented here illustrate the power of NMR in the non-invasive analysis of microbial transformations. Whilst the definitive identification of products requires purification and full structural elucidation. NMR can provide rapid insights into the nature of these reactions and their regulation in vivo. In addition, once the products have been identified NMR methods allow rapid assessment of the effects of genetic and physiological manipulation, and on competing metabolic fluxes with mixed substrates and branched pathways.

Benzene Derivatives↗
PubMed1986

Functional coupling between enzymes of the chromaffin granule membrane.

The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule.

Animals↗
PubMed1986

Electron transfer across the chromaffin granule membrane. Use of EPR to demonstrate reduction of intravesicular ascorbate radical by the extravesicular mitochondrial NADH:ascorbate radical oxidoreductase.

A two-compartment electron paramagnetic resonance system has been developed in which the membrane-impermeable spin probe Ni(en)2+3 is used to selectively eliminate the EPR signal from extravesicular ascorbate radical, such that radicals in intra- and extravesicular compartments can be distinguished. Using this system, we have shown that an increase in ascorbate radical in the extravesicular medium is reflected by an increase in ascorbate radical within resealed chromaffin granule ghosts containing trapped ascorbate but has no effect on radical concentrations inside liposomes containing ascorbate. This indicates that the chromaffin granule membrane contains a component, not present in liposomes, that allows equilibration between the intra- and extravesicular ascorbate/ascorbate radical couples. This component is probably cytochrome b561. We further show that activation of the mitochondrial NADH:ascorbate radical oxidoreductase in the extravesicular medium causes a decrease in intravesicular ascorbate radical in chromaffin granule ghosts but not in liposomes. These data provide direct experimental evidence for the hypothesis that the adrenal medullary mitochondrial NADH:ascorbate radical oxidoreductase could drive the re-reduction of ascorbate free radical generated inside the chromaffin granule by the turnover of dopamine beta-hydroxylase, without the ascorbate radical ever having to leave the granule.

Animals↗
PubMed1979

The exchange of histidine C-2 protons in superoxide dismutases. A novel method for assigning histidine-metal ligands in proteins.

The rates of exchange of the C-2 protons of histidine residues in copper-zinc superoxide dismutase are substantially decreased by metal ion binding. This observation was used to distinguish between ligand and non ligand histidine residues in bovine and yeast copper-zinc superoxide dismutases; the effect was shown to depend only on metal ion co-ordination and not as a consequence of concomitant changes in protein structure. Selective deuteration of the zinc-only proteins at pH (uncorrected pH-meter reading) 8.2 and 50 degrees C resulted in the distinction between copper and zinc ligand resonances in the 1H n.m.r. spectrum of the enzymes. This method is proposed as a generally applicable technique for identifying histidine residues as ligands in metalloproteins.

Animals↗
PubMed1979

Zinc(II) binding to apo-(bovine erythrocyte superoxide dismutase).

The binding of zinc(II) ions to apo-(bovine erythrocytes superoxide dismutase) was studied by 1H n.m.r. spectroscopy. Two zinc(II) ions bind to each subunit of the apoenzyme, and the first has a binding constant at least an order of magnitude larger than the second. The nature of the spectral changes that occur on binding the first zinc(II) ion are interpreted in terms of a change in the structure of the protein around the active site to one very similar to that of the holoenzyme, thus pre-forming the second zinc(II) binding site. The binding of the second zinc(II) ion effects changes in the environment of only those residues involved in its co-ordination.

Animals↗
PubMed1977

Carbon-2 proton exchange at histidine-41 in bovine erythrocyte superoxide dismutase.

The C-2 proton of one histidine residue in bovine erythrocyte superoxide dismutase is shown to be particularly labile. This residue is identified by tritiation, protein digestion and subsequent peptide 'mapping' as histidine-41. A half-life for the exchange of histidine C-2 1H for 2H in 2H2O as solvent, at pD 8.1 and 40 degrees C, is estimated as approx. 9.2h, by 1H nuclear-magnetic-resonance spectroscopy.

Amino Acids↗