Enzymatic Determination of Tissue Dihydrofolate

Abstract

A highly specific and sensitive enzymatic assay for dihydrofolate has been developed. The method involves enzymatic conversion of dihydrofolate to methylenetetrahydrofolate with subsequent stoichiometric entrapment of this folate cofactor into a covalent, ternarv complex with thymidylate synthase and tritiated-fluorodeoxyuridylate. The detection limit in tissue extracts was 0.25 pmoles and reference dihydrofolate was quantitatively recovered in the range 0.25 to 2.00 pmoles by this procedure.     

An Electrochemical Determination of Uric Acid in Sera by a Flow-Through Equipment with and Without Uricase

Abstract

A comparison of an enzymeless direct electrochemical oxidation procedure at a platinum electrode for the determination of uric acid, and an enzyme sensor with immobilized urate: oxygen oxidoreductase (uricase), was performed in flow stream systems. The uricase enzyme electrode is based on the H₂O₂ oxidation current. Both amperometric methods were related to the wall-known photometric uricase-catalase-procedure (UCM) as a reference method. The measured values of both methods are of the first derivatives of current change (dI/dt) due to the electrochemical or electrochemical enzymatic reaction, respectively. The analytical quality of the measurements is characterized by: precision s% within run < 2% day to day < 5% accuracy acceptable (control materials) correlation to reference method r >0.93 analysis rate 80 samples/hr     

Enzyme Electrode Sensor for Hydrogen Peroxide

Abstract

An enzymatic sensor was constructed from an oxygen amperometric sensor on whose surface a dialysis membrane containing covalently bonded catalase was set. Immobilization of the enzyme on the dialysis membrane surface was achieved with 2,4-dichloro-6-methoxy-s-triazine, a derivative of cyanuric chloride, which unlike others of its monosubstituted derivatives ensures some advantages. The time of measurement is less than 12 sec, for the kinetic method of the initial slope and one minute for the steady-state method. The sensor responds linearly to hydrogen peroxide in the concentration range 10^?3 - 10^?5 moles/1. The utilization of this sensor in intermittent and continuous operation in a laboratory enzymatic minireactor is discussed.     

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.     

Amperometric Determination of Lactic Acid. Applications on Milk Samples

Abstract

A hydrogen peroxide electrochemical sensor, coupled with immobilized lactate oxidase and covered with a cellulose acetate dialysis membrane has been applied in flow analysis of lactate in milk samples. The hydrogen peroxide produced by the enzymatic reaction is measured with a platinum electrode polarized at +650 mV versus Ag/AgCl. Milk samples were analyzed and compared with a spectophotometric reference method. The developed procedure is very simple and the short response time allows its use in assaying milk samples on dairy farms.     

Bis-Thiourea Bearing Aryl and Amino Acids Side Chains and Their Antibacterial Activities

Abstract

A series of symmetrical 1,3-bis thiourea 1a–e and 1,4-bis thiourea derivatives 2a–e have been successfully synthesized from the reactions of amines with 3-acetylbenzoyl isothiocyanate and 4-acetylbenzoyl isothiocyanate, respectively. All the synthesized compounds were characterized by FT-IR spectroscopy and ¹H and ¹³C NMR spectroscopy. The compounds were screened for their antibacterial activity by turbidimetric method using gram-negative bacteria (E. coli ATCC 8739) using turbidimetric method. The newly synthesized bis-thiourea derivatives bearing aryl side chains showed good antibacterial activity against E. coli. The effect of the molecular structure of the synthesized compounds on the antibacterial activity is discussed.     

Determination of Particulate Atomospheric Benzoic Acid by Ion Chromatography with Ultraviolet Detection

Abstract

A simple method involving ion chromatography with ultraviolet detection (IC-UV) is applied to the determination of benzoic acid in atmospheric samples. The detection limit is 10 nanograms. Validation and interference studies are described. The application of the IC-UV method to other aromatic acids including nitrophenols is briefly discussed.     

Fast Spectrometric Method of Determining the Activity of Glucose Oxidase

Abstract

A method is described of fast spectrometric determination of glucose oxidase activity based on enzymatic reduction of benzoquinone to hydroquinone and on the measurement of the rate of increase of hydroquinene absorbance at 290 nm. The enzymatic activity is deduced from the initial rate. The method is rapid and sensitive.     

Flow Injection Analysis of Glucose in Human Serum by Chemiluminescence

Abstract

Combining the rigidly controlable conditions of Flow Injection Analysis with detection by chemiluminescence, a method for determination of glucose in serum via enzymatic degradation by glucose in serum via enzymatic degradation by glucose oxidase is described. With a Standard deviation of less than 2% the sampling frequency is 75 samples per hour. Results obtained by the proposed procedure are compared with those acquired by a routine AutoAnalyzer method used at a local hospital. It is shown that any influence on the yield of chemiluminescence due to difference in viscosity of the injected samples effectively can be eliminated.     

Efficient One‐Pot Synthesis of Hydroxyflavanones by Cyclization and O‐Demethylation of Methoxychalcones

An efficient one‐pot method for the synthesis of hydroxyflavanones is described. Methoxychalcones are treated with 36% HBr to afford cyclization and regioselective O‐demethylation products (2a–i) while cyclization and complete O‐demethylation products (3a–e) are obtained in the presence of 45% HI.     

Determination of indole-type phytonutrients in cruciferous vegetables

Abstract

Consumption of cruciferous vegetables has been associated with a low risk of developing cancer. Indole-type phytonutrients, derived from enzymatic hydrolysis of glucobrassicin, exhibit cancer-preventive properties and occur in all vegetables of the Brassicaceae family. A LC-Q-TOF-MS methodology was developed and applied in extracts from seven cruciferous vegetables allowing the rapid determination of indole-3-carbinol, indole-3-carbaldehyde, ascorbigen, indole-3-acetic acid and indole-3-acetonitrile. The novel method described herein, was validated and is characterized by low detection limits and excellent linearity. The simultaneous determination of indole-type phytonutrients in turnip and radish was performed for the first time.     

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.     

Models of biotin coenzyme reactions activated through phosphorylation

Abstract

The reactivity of models of enzymatic carboxylation reactions mediated by biotin coenzyme is presented; a possible role of the requiered ATP molecule for these reactions was investigated.

Several models of biotin activated through phosphorylation - according to the O-phosphobiotin model - are described and their reactivity to wards carboxylating agents discussed.

Models also exhibit phosphoryl transfer properties which may mimic known ATP synthesis from ADP, catalyzed by biotin carboxylases. Possible involvement of the same O-phosphobiotin structure is considered.     

Simultaneous Determination of Carbamazepine,Phenobarbital and Their Major Metabolites in Serum,Brain Tissue and Urine by High-Performane Liquid Chromatographya

Abstract

A high-performance liquid chromatographic method is described for the simultaneous measurement of carbamazepine, phenobarbital and their major metabolites in small samples of serum, brain tissue and urine. This involves solvent extraction, reversed-phase chromatography and ultraviolet detection at 195 nm. Glucuronides in urine are hydrolyzed by enzymatic cleavage with β-glucuranidase. Quantitation is based on the peak-height ratio of the analyte to its internal standard (10, 11-dihydrocarbamaze-pine or p-methylphenobarbital). The results obtained show that the method is precise and reproducible.     

Synthesis of Biological Active Chromene Benzothiadiazole Derivatives

In this article, we describe the synthesis of various substituted Chromene-[1, 3]thiazolo [2, 1, 3] benzothiadiazoles 4a-l by conventional (Method A) and microwave-assisted (Method B) which achieved reductions in reaction times, higher yields, and a cleaner reaction than the conventional method. A comparative study of these two methods has been discussed.     

Catalytic Determination of Molybdenum by Means of the Sodium Perborate/Bromide/Ascorbic Acid Landolt Reaction Using Potentiometric Measurements

Abstract

A new catalytic method for the determination of 0.1-10 ppm molybdenum is described. Molybdenum catalyses the perborate - bromide ascorbic acid Landolt reaction at pH 3.64. The rate is monitored by potentiometry. A calibration graph is based on the ratio of the reaction times for the blank and the sample, (t(o)/t(c), plotted against the concentration of molybdenum. Optimal conditions for the determination are discussed. Also the effect of various ions and ihterferences are described.     

Complexation of Indole-3-acetic Acid with Iron(III): Influence of Coordination on the π-Electronic System of the Ligand

Summary.  The iron(III) complex of indole-3-acetic acid (1) was prepared, and its physicochemical properties, mode of iron(III) coordination, and electronic structure were studied using UV/Vis, diffuse reflectance infrared Fourier transform (DRIFT), and transmission ⁵⁷Fe M?ssbauer spectroscopic techniques. The data obtained provide evidence that iron(III) is not only coordinated by the carboxylic O-donor atom, but also via the conjugated π-electronic system of the pyrrole moiety involving both the non-shared electronic pair of the heteroatom and the C(2)*C(3) double bond. Considering the well-known increased sensitivity of the pyrrole residue in indole derivatives to oxidation as compared to the benzene ring, as well as the formation of a triple complex (peroxidase-1-O₂) proposed for the enzymatic 1 oxidative degradation mechanism involving as a key step the Fe³⁺ → Fe²⁺ transition in the enzyme form as discussed in literature, it is concluded that iron(III) coordination with 1 can influence the redox properties of the pyrrole ring by affecting its π-electronic system. Received September 17, 2000. Accepted (revised) October 31, 2000     

Determination of Aluminium with 8-Hydroxyquinoline-5-Sulfonic Acid in Presence of A Cationic Surfactant by First and Second Derivative Synchronous Fluorimetry

Abstract

An analytical method has been developed for the fluorimetric determination of nanogram amounts of aluminium in solution. The method is based on the reaction of aluminium with B-hydroxyquinoli-ne-5-sulfonic acid in presence of hexadeciltrimethylammonium bromide as a surfactant agent. Synchronous scanned first and second derivative fluorimetry has been employed to increase the sensitivity of the method. The influence of reaction variables as well as instrumental parameters is discussed. The interference of various foreign ions has also been examined and in some cases eliminated or reduced by addition of 1, 10-phenanthroline.     

REACTIONS OF URACILS, 181: REACTION OF 5-FORMYL-1,3-DIMETHYL-BARBITURIC ACID AND 5-FORMYL-1,3,6- TRIMETHYLURACIL WITH ETHOXYCARBONYL- METHYLENETRIPHENYLPHOSPHORANE

Abstract

Ethoxycarbonylmethylenetriphenylphosphorane (2) reacts with 5-formyl-1,3,6-trimethyluracil (1c) in the expected way to afford the acrylate 3c, while with 5-formyl-1,3-dimethylbarbituric acid (1b) a novel phosphorus ylide/phosphorane 4 is obtained (X-ray analysis). The temperature dependent NMR spectra and the mechanism of formation are discussed.     

Purification of Commercially Prepared Bovine Trypsin by Reverse Phase High Performance Liquid Chromatography

Abstract

A reverse phase high performance liquid chromatographic method for the purification of milligram amounts of commercially prepared bovine trypsin is described. Increased specific enzymatic activity is observed in the purified material. The advantages of using purified trypsin in protein digestions as well as the potential application of reverse phase high performance liquid chromatography for enzyme characterization are described.