Biomedical subjects

W J Albery

Publications and source records attributed to W J Albery.

At least 19 recordsLinked to original sources

PubMed1991

Molecular recognition and molecular sensors.

Enzyme-substrate recognition provides a convenient and powerful basis on which to construct molecular sensors. In direct enzyme electrodes the rate of the enzyme reaction is transduced into a current using an electrode made of a conducting organic salt. In vivo microelectrodes designed to measure glucose have been constructed and used in the brain of the freely moving rat. Another strategy is to use enzymes that operate with NADH; the NADH can readily be oxidized on conducting organic salt electrodes. Results for the measurement of micellar equilibria involving bile acids are presented. The packed-bed wall-jet electrode provides a device of greater sensitivity; results for the measurement of femtomoles of acetylcholine obtained by microdialysis from cerebral fluid demonstrate the power of this method. The wall-jet ring-disc electrode can be used in an electrochemical immunoassay again at the femtomole level. Finally, enzyme inhibition can be used to make a sensor for toxic substances such as H2S at the p.p.m. level.

Acetylcholine↗

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↗

PubMed1989

Evolutionary optimization of the catalytic effectiveness of an enzyme.

The kinetic and thermodynamic features of reactions catalyzed by present-day enzymes appear to be the consequence of the evolution of these proteins toward maximal catalytic effectiveness. These features are identified and analyzed (in detail for one substrate-one product enzymes) by using ideas that link the energetics of the reaction catalyzed by an enzyme to the maximization of its catalytic efficiency. A catalytically optimized enzyme will have a value for the "internal" equilibrium constant (Kint, the equilibrium constant between the substrates and the products of the enzyme when all are bound productively) that depends on how close to equilibrium the enzyme maintains its reaction in vivo. Two classes are apparent. For an enzyme that operates near equilibrium, the catalytic efficiency is sensitive to the value of Kint, and the optimum value of Kint is near unity. For an enzyme that operates far from equilibrium, the catalytic efficiency is less sensitive to the value of Kint, and Kint assumes a value that ensures that the rate of the chemical transformation is equal to the rate of product release. In each of these cases, the internal thermodynamics is "dynamically matched", where the concentrations of substrate- and product-containing complexes are equal at the steady state in vivo.

Biological Evolution↗

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

Energetics of enzyme catalysis. I. Isotopic experiments, enzyme interconversion, and oversaturation.

An enzyme-catalyzed interconversion of one substrate, S, and one product P, by an enzyme that exists in two forms E1 and E2 where E1 binds S and E2 binds P, is considered S + E1 in equilibrium E1S in equilibrium E2P in equilibrium E2 + P. Under reversible conditions (where the concentrations of S and P are not far removed from their equilibrium values) it is shown that, in addition to the usual unsaturated and saturated behaviour there exists a third regime at high substrate concentration: the oversaturated region. In this region, the rate-limiting transition state is the interconversion of the unliganded forms of the enzyme: E1 and E2. Expressions for six different experiments involving deuterium, tritium and 14C labels are presented. By considering the results from these experiments, the nature and importance of the enzyme interconversion steps can be elucidated.

Catalysis↗

PubMed1987

Energetics of enzyme catalysis. II. Oversaturation, case diagrams, reversible and irreversible behaviour.

Most enzymes react in vivo under reversible conditions where the substrate and product concentrations are not far removed from equilibrium values. Under these conditions when the concentration of substrate is increased, in addition to the usual unsaturated and saturated behaviour we find a third type of kinetic regime at high substrate concentration-oversaturation. In this regime the rate limiting transition state involves interconversion of free enzyme forms. For a one substrate/one product enzyme, case diagrams can be constructed which depict the kinetic behaviour as a function of substrate and product concentrations. Six different cases are found and are discussed with the relevant free energy profiles. A systematic procedure is described for the investigation and construction of the case diagram.

Catalysis↗

PubMed1986

An amperometric enzyme electrode for monitoring brain glucose in the freely moving rat.

Brain glucose concentration was measured with an amperometric enzyme electrode using glucose oxidase (EC 1.1.3.4) irreversibly adsorbed onto an organic conducting salt. The responses of the electrode and its stability both in vitro and in vivo are described. Parellel changes in brain glucose and blood glucose (measured in samples from an implanted intra-atrial cannula) following injections of insulin are reported.

Animals↗

PubMed1986

Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes.

The interconversion of L- and D-proline catalyzed by proline racemase has been studied. The entire time course of the approach to equilibrium has been followed. After a short time the product concentration is significant, and the reaction runs under reversible conditions. As the total substrate concentration is increased, the system moves from the unsaturated regime into the saturated regime. At very high substrate levels under the reversible conditions used, the rate constant for substrate racemization falls, as the system moves into the "oversaturated" regime. Here, the net rate of the enzyme-catalyzed reaction is limited by the rate of return of the free enzyme from the form that liberates product back to the form that binds substrate. The results are analyzed in terms of the simple mechanism (table; see text) and illustrate the additional information that is available from reactions studied under reversible conditions. In the unsaturated region the value of the second-order rate constant kU (equivalent to kcat/Km) is 9 X 10(5) M-1 s-1 in each direction. In the saturated region, kcat = kcat = 2600 s-1 and Km = 2.9 mM. In the oversaturated region, the rate constant kO is 81 M s-1. The substrate concentration at which unsaturated and saturated terms contribute equally is 2.9 mM, and the substrate concentration at which saturated and oversaturated terms contribute equally is 125 mM.

Amino Acid Isomerases↗

PubMed1986

Energetics of proline racemase: tracer perturbation experiments using [14C]proline that measure the interconversion rate of the two forms of free enzyme.

Proline racemase exists in two states, one of which binds and isomerizes L-proline and the other of which binds and isomerizes D-proline. In the enzyme-catalyzed racemization of proline at high substrate concentrations, the interconversion of the two forms of the free enzyme becomes rate limiting. The tracer perturbation method of Britton (1966, 1973) vividly demonstrates the kinetic importance of this enzyme interconversion under oversaturating conditions and allows an estimate of the rate constant for this reaction of 10(5) s-1. It is further shown that the enzyme is bound state saturated and the peak-switch concentration, Cp, is 125 mM. At substrate concentrations higher than 125 mM the enzyme becomes oversaturated, and the reaction rate is limited by the transition state for the interconversion of two forms of the free enzyme. It seems likely that the two free enzyme forms differ only in the protonation states of the acidic and basic groups at the active site.

Amino Acid Isomerases↗

PubMed1986

Energetics of proline racemase: transition-state fractionation factors for the two protons involved in the catalytic steps.

The isotope effects for the interconversion of L-proline and D-proline, catalyzed by proline racemase, have been determined in the saturated region with both [2-2H]proline and [2-3H]proline. The deuterium fractionation factors for each of the protons in flight have been obtained from two kinds of experiment: by measuring the rate of racemization of one [2-2H]proline enantiomer as it racemizes into an equilibrated pool of unlabeled proline and by measuring the deuterium content of a proline sample at the optical rotation maximum that occurs when an equimolar mixture of one deuterium-labeled enantiomer and the other unlabeled enantiomer runs to equilibrium. The tritium fractionation factors for each of the protons in flight have been determined from measurements of the rate of loss of tritium to the solvent as one [2-3H]proline enantiomer runs to equilibrium. Good agreement is found among the fractionation factors determined by each method. The deuterium fractionation factors for the two protons are not identical: that for the proton derived from L-proline is 0.375 and that for the proton derived from D-proline is 0.44. This difference has been confirmed by a double-competition experiment in which the optical rotation of a mixture of DL-[2-2H]proline and unlabeled DL-proline is followed with time. The rotation (initially zero) passes through a maximum, from which the ratio of the two fractionation factors (0.86) is obtained. These data, coupled with the equilibrium fractionation factor for the 2-position of proline (which has been determined to be 1.17), provide the transition-state factors for each of the in-flight protons, and delineate the nature of the transition state(s) for the enzyme-catalyzed racemization.

Amino Acid Isomerases↗

PubMed1986

Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance.

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.

Amino Acid Isomerases↗

PubMed1986

Energetics of proline racemase: fractionation factors for the essential catalytic groups in the enzyme-substrate complexes.

The fractionation factors of protons bound to the essential catalytic groups in proline racemase have been determined by comparison of the time courses of two competitive deuterium washout experiments. The rate of achievement of the maximum perturbation in the optical rotation has been measured in the oversaturated region (that is, at high substrate concentrations) under two conditions: in the first, we start with an equimolar mixture of deuterated substrate S' and of unlabeled product P; in the second, we again start with equal concentrations of substrate and product, but the concentration of the deuterated material S' is less than 20% that of S. The different concentrations of deuterated substrate produce different levels of deuteration of the enzyme's catalytic groups, the kinetic consequence of which allow the fractionation factors of these enzymic groups to be determined. The observed values for the fractionation factors of the enzyme's groups of 0.55 +/- 0.1 are only consistent with these groups' being thiols. This conclusion is supported by results of measurements of the solvent isotope effect determined in the unsaturated regime. These findings confirm the earlier suggestion of Abeles and his group that two cysteine residues mediate the catalysis of proline racemization by this enzyme.

Amino Acid Isomerases↗

PubMed1986

Energetics of proline racemase: rates, fractionation factors, and buffer catalysis in the oversaturated region. Nature of the interconversion of the two forms of free enzyme.

To probe the nature of the interconversion of the two unliganded forms of proline racemase, a number of experiments have been performed under oversaturating conditions where the rate of the enzymic reaction is mainly limited by the rate of this interconversion. Competitive deuterium washout experiments, where an equimolar mixture of D- and L-proline (in which some or all of one enantiomer is specifically deuterated at the 2-position) is allowed to reach chemical and isotopic equilibrium mediated by the enzyme, have been followed in four ways. The size and the rate of achievement of the maximum perturbation in the optical rotation have been measured, the deuterium content of the substrate at this maximum has been determined, and the final approach to equilibrium after the perturbation maximum has been followed. Further, the enzyme-catalyzed rate of tritium loss from [2-3H]proline has been established. Finally, it has been shown that the enzyme interconversion reaction is catalyzed by several buffers (such as ammonium, hydrazinium, and hydrogen sulfide). These data are discussed in terms of Marcus' theory, which allows a rather detailed picture of the mechanism of free enzyme interconversion to be drawn. This process nicely parallels the mechanism of the enzyme-catalyzed interconversion of the proline enantiomers, and it is evident that substrate racemization (with the concomitant switch of the enzyme-bound protons) is mirrored by the water-mediated switch of the enzyme-bound protons that effects the interconversion of the free enzyme forms. The results favor a stepwise reaction for the interconversion of the free enzyme forms in which a proton is abstracted from a bound water molecule to give a reaction intermediate having a hydroxide ion bound to the diprotonated form of the enzyme.

Amino Acid Isomerases↗

PubMed1986

Energetics and mechanism of proline racemase.

The results from the previous six papers are collated so as to allow the construction of the complete free energy profile for the reaction catalyzed by proline racemase. This profile includes the step that involves the isomerization of the two forms of free enzyme, which can become rate limiting at very high substrate levels (in "oversaturation"). The mechanism of the reaction has been defined, the results being best accommodated by a route that involves a transition state or unstable intermediate in which the proline carbanion is flanked by the two catalytic thiols of the enzyme.

Amino Acid Isomerases↗

PubMed1984

Voltammetric carbon paste electrodes monitor uric acid and not 5-HIAA at the 5-hydroxyindole potential in the rat brain.

Changes in the height of peak 2 obtained using linear sweep voltammetry and carbon paste electrodes chronically implanted in discrete brain regions of the unrestrained rat were measured under a variety of conditions; in the past this peak has been attributed to the oxidation of 5-hydroxyindoleacetic acid (5-HIAA). Unilateral 5,7-dihydroxytryptamine (5,7-DHT) lesions of the medial forebrain bundle reduced the 5-HIAA content of the striatum and hippocampus to 10% of the unlesioned side, but did not alter the height of peak 2 recorded in these regions. In contrast, microinfusion of uricase beside striatial electrodes reduced the height of peak 2 by 96%; systemic amphetamine-induced increases in the height of the peak were also prevented by this enzyme. These results indicate that uric acid, and not 5-HIAA, is mainly responsible for peak 2, and that changes in the height of this peak reflect changes in the extracellular concentration of uric acid.

Animals↗

PubMed1984

Electrochemical aspects of intravascular oxygen transducers.

The factors which limit the useful lifetime of miniaturized electrochemical oxygen transducers for use in neonatal intensive care are described. These factors arise from the use of the conventional silver-silver chloride reference electrode. Four possible means of improving the useful lifetime are described, namely the use of ion-permeable membranes, guard electrodes, potential-time programmes and nickel(II) hydroxide reference electrodes.

Blood Gas Analysis↗