HPLC Determination of Hepatic Cholesterol 7α-Hydroxylase Activity Using Immobilized Cholesterol Oxidase and Chemiluminescence Detection

Abstract

This paper describes an HPLC method for the determination of cholesterol 7α-hydroxylase activity, at high or low activity levels, that is sensitive and specific for 7α-hydroxycholesterol. The method relies on the generation of hydrogen peroxide by oxidation of 7α-hydroxycholesterol using the enzyme cholesterol oxidase which has been immobilized on porous glass beads. The hydrogen peroxide is subsequently detected by chemiluminescence generated by reaction of peroxide with bis-(2, 4, 6-trichlorophenyl)-oxalate (TCPO), a commonly used chemiluminescence reagent specific for peroxides. In the procedure, sample preparation is limited to extraction of the incubation mixture and injection of the concentrated extract.     

In vitro determination of diamine oxidase activity in food matrices by an enzymatic assay coupled to UHPLC-FL

Intestinal diamine oxidase (DAO) acts as a protective barrier against exogenous histamine. A deficit of DAO activity can lead to the appearance of histamine intolerance, a clinical condition that may be treated by a low-histamine diet and oral DAO supplementation to enhance intestinal histamine degradation. As sources of DAO, porcine kidneys and certain legume seedlings are suitable components for the formulation of a DAO supplement. The aim of this work was to develop a rapid and reliable methodology for the in vitro determination of DAO activity in food matrices based on an enzymatic assay coupled to UHPLC-FL. The proposed method showed a satisfactory linearity and sensitivity and provided a relative standard deviation lower than 3%, guaranteeing method precision, and a mean recovery greater than 99% both for lyophilized pea sprouts and porcine kidney protein extracts. A high specificity is a key attribute of this method due to the use of histamine as the reaction substrate and the direct quantification of its degradation. Moreover, the lack of interference of catalase and hydrogen peroxide is another advantage in comparison with previously published methods. Lyophilized pea sprouts showed the greatest histamine-degrading activity (0.40 ± 0.01 mU/mg), followed by porcine kidney protein extracts (0.23 ± 0.01 mU/mg) and commercial DAO supplements (0.09 ± 0.06 mU/mg). This technique could be used as a tool to validate the DAO activity of food matrices of potential interest for the treatment of histamine intolerance.

     

Determination of Acetylcholine and Choline in Rat Brain Tissue by Liquid Chromatography/Electrochemistry Using an Immobilized Enzyme Post Column Reactor

Abstract

Following separation by conventional LC, acetylcholine and choline are converted to hydrogen peroxide in a packed bed reactor consisting of covalently bound acetylcholinesterase and choline oxidase. Hydrogen peroxide, the ultimate enzymatic product, is detected amperometrically at a platinum electrode thin-layer cell. This method is simple and highly sensitive to the detection of acetylcholine and choline, with detection limits of about 100 femtomoles. The immobilized enzyme columns were stable for at least 60 days and conserved precious and expensive supplies of enzyme relative to continuous addition schemes. The apparatus is generally applicable to other enzymatic reactions which yield electroactive products.     

Ultrasound assisted biobleaching of cotton

In this study, the effect of ultrasound on the activity of the glucose oxidase (GOx) enzyme for bleaching of the cotton fabrics was investigated. Hydrogen peroxide generation with the GOx enzyme from glucose was carried out under ultrasonic homogenizer (UH) and ultrasonic bath support. The aim of using ultrasonic support was to increase the yield of the enzyme reactions. The enzymatically generated hydrogen peroxide was used for bleaching of cotton fabrics. The bleaching process was performed at 90 °C and pH 11 (with NaOH) for 60 min, followed by rinsing at 70 and 50 °C then cold washing. The whiteness degrees of the cotton samples that were bleached by the generated peroxide were compared to the whiteness degrees of the conventionally bleached cotton fabrics. Sufficient whiteness degrees in cotton fabrics could be obtained by enzymatically generated hydrogen peroxide by UH support. The initial whiteness degree of the cotton fabric was 59.9 Stensby degrees; the whiteness was increased to 75.6 Stensby degrees by the GOx enzyme under UH support where the conventional bleaching process yielded a whiteness value of 76.7 Stensby degrees. For efficient cotton bleaching by the GOx enzyme, UH support contributed to the concentration of enzymatically generated hydrogen peroxide by the GOx enzyme. Bleaching of cotton by the GOx enzyme was approved as a more environmentally friendly process compared to the conventional bleaching method in respect of the results of chemical oxygen demand tests.     

Electroanalysis of D-Amino Acid Oxidase and Its Interaction with Hydrogen Peroxide

Abstract

We have first obtained the direct electrochemistry of D-amino acid oxidase with a polyethylenimine modified pyrolytic graphite electrode and the electrochemical response related to the catalytic reaction to the substrates. Further studies reveal that the enzyme may exhibit different substrate specificity. Taking D-serine as a model, we have also presented an electrochemical method to detect this amino acid and have studied the effect of hydrogen peroxide on the catalytic activity of this enzyme. It is found that hydrogen peroxide can lower the enzymatic activity of this enzyme.     

Development and evaluation of a highly sensitive rapid response enzymatic nanointerfaced biosensor for detection of putrescine

Putrescine (1,4-diaminobutane) a biologically active diamine has been found to be a valuable analyte for several clinical and analytical purposes. The present work deals with diamine oxidase immobilized on iron oxide nanoparticles for quantifying the amount of putrescine produced, by the decarboxylation of ornithine, which is converted into hydrogen peroxide by the enzyme diamine oxidase (DAO). This reaction can be quantified using electrochemical techniques, which forms the basis of this work. Iron oxide (Fe(3)O(4)) nanoparticles, synthesized using thermal co-precipitation, were chosen for immobilization of DAO due to its simple preparation procedure, high surface area and cost-effectiveness. The size of the particles was in the range of 25-35 nm and the enzyme was linked covalently by carbodiimide activation and confirmed using FT-IR. For detecting the hydrogen peroxide released in the reaction, a glassy carbon-working electrode coated with enzyme linked iron oxide nanoparticles was poised at +0.4 V versus an Ag/AgCl reference electrode and a platinum wire was used as the counter electrode. A step-wise increase in current was observed and linearity was obtained in the range of 2-8 nM, with 0.65 nM as the minimum detection limit and the response time was found to be 0.3 seconds. Ascorbic acid, a common interfering molecule in biological samples, did not interfere with the measurements indicating the high degree of specificity of the diamine oxidase-based nano-interfaced biosensor.     

Activity staining of plasma amine oxidase after polyacrylamide gel electrophoresis and its application to natural inhibitor screening

Plasma amine oxidase (plasma AO, EC 1.4.3.6) is a copper-containing AO which converts benzylamine (BZ) to benzaldehyde, generating hydrogen peroxide and ammonia. The peroxidase was used as an ancillary enzyme to couple hydrogen peroxide to 3-amino-9-ethylcarbazole (AEC) to achieve plasma AO activity after electrophoresis on native polyacrylamide gels. It was confirmed that plasma AO is inhibited by semicarbazide but neither by clorgyline nor by deprenyl. We also used plasma AO activity staining for the screening of natural inhibitors. This fast and sensitive method can be used in the process of plasma AO purification, characterization, and inhibitor screening.     

Development of a diamine biosensor

An amperometric diamine sensor is developed for clinical applications in diagnosis of bacterial vaginosis (BV). The sensor is based on crosslinked putrescine oxidase (PUO) which catalyzes the conversion of diamines (mainly putrescine and cadaverine) to products including hydrogen peroxide. The hydrogen peroxide is detected anodically at platinum electrode polarized at 0.5 V versus Ag/AgCl. Platinum-plated gold electrodes used as a substrate for the sensor construction, are batch-fabricated on a flexible polyimide foil (Kapton(R), DuPont). A three-electrode cell configuration is used in all amperometric measurements. The sensor construction is based on three layers: an inner layer to reject the interference effect of oxidizable molecules, an outer diffusion controlling layer, and in addition, an enzyme middle layer. The enzyme layer was immobilized by crosslinking PUO with bovine serum albumin (BSA) using glutaraldehyde (GA). An optimization study of the enzyme solution composition was carried out. With the optimized enzyme layer, the biosensor showed a very high sensitivity and fast response time of ca. 20 s. The sensor has a linear dynamic range from (0.5-300 muM) for putrescine that covers the expected biological levels of the analyte. Details on sensor fabrication and characterization are given in the present work.     

Application ofo-Hydroxyphenylfluorone as a Fluorogenic Indicator to the Determination of Hydrogen Peroxide and Oxidase Substrates Based on the Catalytic Effect of Hemin

A highly sensitive fluorescence quenching method has been developed for selective determination of hydrogen peroxide based on the catalytic effect of hemin on theo-hydroxyphenylfluorone (a new fluorogenic substrate) and hydrogen peroxide system. Under optimum conditions, the calibration graph was linear over the range 0–1.0 × 10⁻⁶mol/liter hydrogen peroxide, with a limit of detection of 8.0 × 10⁻⁹mol/liter in a 10-min reaction period. It can easily be incorporated into the determination of biochemical substances that produce hydrogen peroxide under catalytic oxidation by their oxidase. This possibility has been tested for the determination of glucose in human sera as an example.     

Multipurpose electrode with different enzyme systems bound to collagen films

Selective, multipurpose electrodes have been developed from the previously described glucose electrode based on amperometric detection of hydrogen peroxide. Several single or multi-enzyme systems, including galactose oxidase, cholesterol oxidase, glucoamylase with glucose oxidase, and invertase with glucose oxidase, can be covalently bound to collagen membranes and attached to a platinum anode for monitoring the hydrogen peroxide generated. The probes allow fast and sensitive measurements of galactose, free cholesterol and maltose. Analogous electrodes are convenient for the assay of sucrose and lactose, with lower sensitivity. For disaccharide measurements, a comparative study of membranes produced by random co-immobilization, stacking of membranes and asymmetric coupling is reported. Asymmetric coupling improved the electrode performances in every case. One enzyme membrane is readily replaced by another in the electrode construction, and the sensors can be used for hundreds of assays.     

A new staining method for the detection of activities of H2O2-producing oxidases on gels and blots using cerium and 3,3'-diaminobenzidine.

Cerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown-black precipitate by the subsequent reaction with 3,3'-diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecificity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms.     

Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

A fast, simple and sensitive bioelectrochemical magnetic immunosensing method is developed to monitor a potential insecticide biomarker, trichloropyridinol (TCP), in environmental sample. A magnet/glassy carbon (MGC) working electrode was used to accumulate immunocomplex associated magnetic beads and separate free and unbound reagents after liquid phase competitive immunoreaction among TCP antibody coated magnetic beads, TCP analyte and horseradish peroxidase (HRP) labeled TCP. The activity of HRP tracers was monitored by square‐wave voltammetry (SWV) by scanning electrocactive enzymatic product in the presence of 3,3′,5,5′‐tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB‐H₂O₂) substrate solution. The electrochemical signal of enzymatic product was greatly enhanced by dual accumulation events: magnetic accumulation of enzyme tracers bound magnetic beads and constant potential accumulation of enzymatic product. The voltammetric characteristics of substrate and enzymatic product were investigated, and the parameters of SWV analysis and immunoassay were optimized. Under the optimal conditions the immunosensor was used to measure as low as 5 ng L^?1 (ppt) TCP, which is 50‐fold lower than the value indicated by the manufacture of the TCP RaPID Assay kit (0.25 μg/L, colorimetric detection). The performance of the developed immunosensing system was successfully evaluated with river water samples spiked with TCP, indicating this convenient and sensitive technique offers great promise for decentralized environmental application. This technique could be readily used for detection of other environmental contaminants by developing specific antibodies against the contaminants and are expected to open new opportunities for environmental monitoring and public health.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

N,N-diethylaniline as hydrogen donor for determination of hydrogen peroxide catalyzed by metalloporphyrins as enzyme mimetic of peroxidase

Various metalloporphyrins have been used as a catalyst instead of the peroxidase for the determination of hydrogen peroxide by formation of a dye from N,N-diethylaniline (DBA) and 4-aminoantipyrine. The difference of relative catalytic activity was investigated between enzyme and enzyme mimetics. FeT(4-TAP)P [5, 10, 15, 20-tetrakis (4-trimethyl-ammoniumphenyl)-21H, 23H-porphine] was shown as the best enzyme mimetic for horseradish peroxidase (HRP) among metalloporphyrins tested. 0 to 7.0 × 10^–5 mol/L hydrogen peroxide was determined with good accuracy and reproducibility, and giving recovery of 99.7–100.7%. DEA was certified as a sensitive color reagent in enzyme mimetic assay of hydrogen peroxide, with the apparent molar absorptivity for hydrogen peroxide was 1.37 × 10⁴ L/mol·cm.     

Phosphate-sensitive enzyme electrode: a potential sensor for environment control

A highly sensitive enzyme electrode was designed for the assay of phosphate ions. For this purpose, a bienzyme membrane with co-immobilized nucleoside phosphorylase and xanthine oxidase was used with a platinum amperometric electrode for the detection of enzymatically generated hydrogen peroxide. A detection limit of 10^?7 M was obtained and phosphate assays could be easily performed in the range 0.1–10 μM, which is of interest in the control of water pollution.     

Development of a highly sensitive fluorescence probe for hydrogen peroxide

Hydrogen peroxide is believed to play a role in cellular signal transduction by reversible oxidation of proteins. Here, we report the design and synthesis of a novel fluorescence probe for hydrogen peroxide, utilizing a photoinduced electron transfer strategy based on benzil chemistry to control the fluorescence. The practical value of this highly sensitive and selective fluorescence probe, NBzF, was confirmed by its application to imaging of hydrogen peroxide generation in live RAW 264.7 macrophages. NBzF was also employed for live cell imaging of hydrogen peroxide generated as a signaling molecule in A431 human epidermoid carcinoma cells.     

Amperometric assay for collagenase

A sensitive and rapid amperometric assay for collagenase has been developed. The substrate for the assay is glucose oxidase covalently linked to insoluble collagen with dimethylsuberimidate. The collagenase cleaves the insoluble collagen-glucose oxidase conjugate into smaller, soluble fragments that have glucose oxidase activity. That activity is proportional to the collagenase activity hydrolyzing the insoluble conjugate. In the absence of collagenase, no glucose oxidase activity is found in the soluble phase. Glucose oxidase activity was assayed by measuring amperometrically the rate at which hydrogen peroxide is produced. The kinetics follow that proposed for a soluble enzyme acting on an insoluble substrate.     

Highly sensitive detection of l-glutamate by on-line amperometric micro-flow analysis based on enzymatic substrate recycling

A highly selective and sensitive on-line monitoring system is proposed for amperometric assay of trace amounts of l-glutamate. The system includes a microdialysis probe, immobilized enzyme reactor, and poly(1,2-diaminobenzene)-coated platinum electrode. The enzyme reactor prepared by the co-immobilization of l-glutamate oxidase and glutamate dehydrogenase are here employed to enhance the sensitivity of l-glutamate as an on-line amplifier based on the substrate recycling. The l-glutamate in the dialysate from the probe are recycled enzymatically during passage through the reactor in the presence of sufficient amounts of NADH and oxygen to produce a large amount of hydrogen peroxide, which is detected if selectively at a downstream poly(1,2-diaminobenzene)-coated platinum electrode without interference from oxidizable species such as l-ascorbate in the sample and NADH added to the carrier buffer. The cycle is also initiated with 2-oxoglutarate, and so saccharopine dehydrogenase reactor is positioned in series before the amplifier reactor to remove 2-oxoglutarate in the dialysate. By the present method, l-glutamate is selectively assayed with a 160-fold increase in sensitivity compared with the unamplified responses. The detection limit is 0.5x10(-7) M of l-glutamate.     

Simultaneous Determination of Glucose and L‐Glutamate Using a Capillary Enzyme Reactor with Electrochemical Detection

An electrophoresis capillary that incorporates two enzymes for the simultaneous determination of glucose and L ‐glutamate is described. The enzymes deposited along the separation capillary walls react with their respective substrate as they are separated during the electrophoresis to produce hydrogen peroxide that is detected by amperometry as the hydrogen peroxide zone emerges from the end of the capillary. Even though both enzyme reactions produce a common product, hydrogen peroxide, the hydrogen peroxide produced by each enzyme reaction stays in narrow zones that migrate the length of the capillary at different rates. The rate of migration for the individual H₂O₂ zones is consistent with the expected mobility of neutral glucose and of anionic L ‐glutamate, respectively. This property allows each enzyme substrate to be characterized in a single experiment and in the presence of other electroactive substances.     

Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing

Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.