Indirect Flow-Injection Assays for Glucose-6-Phosphate Dehydrogenase/Glucose-6-Phosphate and Malate Dehydrogenase/L-Malate Using Immobilized Bacterial Luciferase

Abstract

Flow-injection procedures for the indirect determination of glucose-6-phosphate dehydrogenase, glucose-6-phosphate, malate dehydrogenase and L-malate are described. They are based on a coupled reaction with bacterial luciferase and oxidoreductase co-immobilized on cyanogen bromide activated Sepharose 4B. the limits of detection are 0.015 pmol for glucose-6-phosphate dehydrogenase, 0.03 pmol for malate dehydrogenase, 10^?8 mol^?1 for glucose-6-phosphate and 10^?6 mol l^?1 for L-malate. the reproducibility is less than 5% relative standard deviation (n=5) for all assays and the sample throughput is 60 h^?1.     

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.     

Determination of glucose in alcoholic beverages by flow injection with two internally coupled injection valves and an enzyme reactor

Two new flow configurations are described for the determination of a component in samples for which the matrix provides varying blank values. They are applied to the determination of glucose in alcoholic beverages, by using immobilized hexokinase and glucose-6-phosphate dehydrogenase, and spectrophotometric determination of the NADPH formed.     

Sequential determination of glucose and fructose in foods by flow-injection analysis with immobilized enzymes

A sequential method for determination of glucose and fructose involving the use of enzymes (hexokinase and glucose-6-phosphate dehydrogenase) immobilized on controlled-pore glass is proposed. The flow is selected so as to determine glucose or both sugars by fluorimetric determination of the NADH formed. The method is applied to the determination of these compounds in fruit juices, yoghourt and dessert powder with good results.     

Efficiency of two enzymes immobilized to the same surface and acting in sequence

Phosphoglucomutase and glucose-6-phosphate dehydrogenase were immobilized to s-triazine trichloride activated cellulose. The optimal conditions for binding the immobilized enzymes were determined and the kinetic and physical properties were investigated.The final ratio of the two enzymes immobilized to the surface was determined by the physical properties of the enzymes as well as by the ratio of the enzymes present in the attachment solution. The immobilized enzymes were found to retain at least 60% of the original activity for at least 40 days when stored at 4°C and in the presence of substrates and cofactors. Immobilized phosphoglucomutase and glucose-6-phosphate dehydrogenase also were much more stable at 58°C, retaining 28 and 13% of the original activity, respectively, after 75 min. The apparentK _m’s were 5.4 to 1.5 times higher for immobilized phosphoglucomutase and glucose-6-phosphate dehydrogenase, respectively than for the soluble enzymes.     

Enzyme Electrode for Fructose,Glucose-6-Phosphate and ATP Determination

Abstract

A bienzyme electrode for glucose-6-phosphate and ATP based on the sequential reactions of coimmobilized glucose-6-phosphate dehydrogenase and hexokinase and for fructose using the competition with glucose has been developed. Electrochemical indication is performed using air-oxidation of reduced N-methylphenazinium ion. The sensor responds linearly to fructose up to 3 mM with a lower detection limit of 0.3 mM.     

Cyclophosphate

The phosphorylase assay is performed in the direction from glycogen to glucose-1-phosphate by coupling with the glucose-6-phosphate dehydrogenase reaction. Using this test, some properties of the activation mechanisms in liver and muscle by cyclophosphates are compared and the results discussed. A large number of cyclophosphates is tested in both systems. Molecular variations in the cyclophosphates shift the activation curves equally in both systems. All cyclophosphates activate the system to an equal degree.     

Coimmobilization of a redox enzyme and a cofactor regeneration system

The coimmobilization of nitrobenzene nitroreductase and glucose-6-phosphate dehydrogenase in silica particles enables the continuous conversion of nitrobenzene to hydroxylaminobenzene with NADPH recycling.     

CHANGES IN SUPEROXIDE DISMUTASE, CATALASE AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITIES OF RABBIT ALVEOLAR MACROPHAGES: INDUCED BY POSTNATAL MATURATION AND/OR IN VITRO HYPEROXIA

Abstract— The total superoxide dismutase activity of rabbit alveolar macrophages (AM) increased twofold during the first postnatal week. This increase in superoxide dismutase activity was primarily mitochondrial and paralleled the increase in the number of mitochondria in these cells which has been previously reported. The superoxide dismutase activity of AM in culture for 18 h was significantly increased by hyperoxia; catalase activity in hyperoxic conditions was slightly adversely affected; glucose-6-phosphate dehydrogenase activity was increased but not significantly over control values. Hyperoxic cultures beyond 42 h decreased the total number of viable cells and the superoxide dismutase activity expressed per viable cell; the disappearance of catalase and glucose-6-phosphate dehydrogenase activities in this period, however, was more rapid in control alveolar macrophages than those under hyperoxia.     

Homogeneous enzyme-linked competitive binding assay for biotin based on the avidin-biotin interaction

A homogeneous enzyme-linked competitive-binding assay for biotin with glucose-6-phosphate dehydrogenase (G6PDH), is described. This assay is based on the interaction between a G6PDH/biotin conjugate with avidin, a natural binder for biotin. In the absence of biotin in the assay mixture, this interaction results in 100% inhibition of the enzyme conjugate. In the presence of biotin, the enzymatic activity of the conjugate is regained in an amount related to the concentration of the vitamin in the sample. Extremely steep, gate-like dose/response curves, attributable to the relative binding affinities of avidin for biotin and the conjugate, are observed. The detection limits of the system vary with the amounts of avidin and enzyme/biotin conjugate used. The method is rapid and sensitive and is evaluated for the direct determination of biotin in vitamin tablets.     

Determination of molecular mass, Stokes' radius, frictional coefficient and isomer-type of non-denatured proteins by time-dependent pore gradient gel electrophoresis

Molecular mass, Stoke's radius, frictional coefficient and isomer-type of non-denatured proteins can be obtained by time-dependent gradient gel electrophoresis by evaluating the resulting data using a two-step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non-denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from current-year needles of spruce. The resulting data as well as those which were calculated for esterase (EC 3.1.1.1), glutamate dehydrogenase (EC 1.4.1.4), isocitrate dehydrogenase (EC 1.4.1.42), and shikimate dehydrogenase (EC 1.1.1.25) are in accordance with those reported in the literature. The method described may be applied to various scientific areas such as genetics or environmental pollution. It could be shown here that current-year needles of injured spruce (damage class 3) contained two more peroxidase isozymes and one more glucose-6-phosphate dehydrogenase isozyme than those from non-injured trees. These differences may mark two genotypes of spruce of different susceptibilities towards present-day air and soil pollutants.     

Micro enzymatic assay coupled to capillary electrophoresis via liquid junction

Summary A system for capillary electrophoresis combined with enzymatic assay has been evaluated for the two enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Instrumentation included a post-column reactor coupled to the separation capillary by a liquid junction. A technique for generating a substrate solution flow into the reactor by utilizing two high voltage supplies is proposed. This method offers a high degree of freedom in optimizing the separation and enzymatic reaction conditions individually. Possibilities for improving the enzymatic assay sensitivity were also examined.     

Liquid Chromatography with Electrochemical Detection of Phenol and NADH for Enzyme Immunoassay

Abstract

Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.     

Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L·h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L·h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (μ) decreased 82% from 0.045 to 0.008 h⁻¹ when OTR changed from 12.6 to 8.4 mmol/L·h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.     

EFFECT OF LIGHT ON OSCILLATIONS OF ENZYME ACTIVITY DURING PHOTOMORPHOGENESIS IN CHENOPODIUM RUBRUM L.

An approach to determine the photomorphogenic effect of light (white or continuous far-red) on the development of rhythmic enzyme activity in Chenopodium rubrum L. is described. Previous results, obtained from mature seedlings grown in white light, demonstrated stable oscillations with periods ranging between 12 and 15 h for all of the enzymes tested. The present results, obtained during deetiolation, were complicated by the presence of a higher frequency component with a period of about 6 h. When the various oscillating components were defined, the analysis showed: (1) the enzymes of the Krebs cycle (malate and isocitrate dehydrogenase), the closely associated glutamate dehydrogen-ase, and the glycolytic pathway ((NAD) glyceraldehyde-3-phosphate dehydrogenase) had a dominant period in the range of 12–15 h, (2) those of the oxidative pentose phosphate pathway were either weakly circadian (glucose-6-phosphate dehydrogenase) or apparently arhythmic (6-phosphogluconate dehydrogenase), (3) the (NADP) glyceraldehyde-3-phosphate dehydrogenase from the Calvin cycle was circadian when kept in continuous darkness but becomes 15 h when placed in light, and (4) only the Calvin cycle enzyme is affected by light in the level of its activity and in its oscillatory behavior.     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

Fluorometric assay of ultramicro quantities of glucose with somogyi filtrate and hexokinase

A procedure is described for the determination of glucose in 3 μl of serum or plasma. It is based on the phosphorylation of glucose with ATP mediated by hexokinase. The TPNH generated with glucose-6-phosphate dehydrogenase is measured fluorometrically.Sensitivity is greatly improved over the corresponding procedure using UV absorption for the determination of NADPH. Bilirubin does not interfere. Interference from hemolysis is eliminated by precipitation with Zn(OH)₂ Blood taken with fluoride can be utilized. Fluoride interference is removed with the Zn(OH)₂ and with excess Mg in the substrate. Positive interference by fructose is negligible with some enzyme preparations but is appreciable with others.The results correlate well with those obtained with an ortho toluidine method, after protein precipitation with trichloroacetic acid. The method is especially useful in the Pediatric Laboratory.     

Homogeneous Amperometric Immunoassay for Digoxin

Abstract

A homogeneous spectrophotometric EMIT immunoassay for the measurement of digoxin has been converted to an amperometric immunoassay format capable of analysing digoxin in diluted serum. NADH produced by glucose-6-phosphate dehydrogenase (G6PDH) labelled digoxin was detected amperometrically by its rapid, efficient oxidation at platinized activated carbon electrodes (PACE) poised at +150 mV vs Ag/AgCl. The new amperometric procedure resulted in a simplified assay protocol with a decreased overall assay time, enhanced NADH detection and minimal sample pretreatment.     

Use of the thermostable glucose-6-phosphate dehydrogenase as a label enzyme in steroid enzyme immunoassays.

A thermostable glucose-6-phosphate dehydrogenase was assessed as a label enzyme in a testosterone enzyme immunoassay system. Enzyme labeling of a steroid with the enzyme was carried out by the N-succinimidyl ester method. A dose-response curve with a satisfactory sensitivity could be obtained by the use of the enzyme-labeled antigen. This enzyme should be useful in hapten enzyme immunoassays because of its relatively high stability.     

Response surface examination of the relationship between experimental conditions and product distribution in electrophoretically mediated microanalysis

This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions.