A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H₂O₂. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 μl of serum at concentrations ranging from 0.02 to 1.5 mm. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mm. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.     

Production of an aromatic aldehyde oxidase by Streptomyces viridosporus

Streptomyces viridosporus strain T7A, when grown in liquid media containing yeast extract and aromatic aldehydes, oxidized the aromatic aldehydes to the corresponding aromatic acids. Benzaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, and protocatechualdehyde were catabolized further via the -ketoadipate and gentisate pathways. Dehydrodivanillin, isophthalaldehyde, salicylaldehyde, syringaldehyde, terephthalaldehyde, vanillin, and veratraldehyde were oxidized only as far as the corresponding aromatic acids. Phthalaldehyde and aliphatic aldehydes were not oxidized. The aromatic aldehyde oxidase, which was produced by cultures grown in either the presence or absence of aromatic aldehydes, was partially purified by ammonium sulfate precipitation and ion-exchange chromatography. It consumed molecular oxygen, oxidized aromatic aldehydes to aromatic acids, and produced hydrogen peroxide all in equimolar amounts.Paper no. 81515 of the Idaho Agricultural Experiment Station     

A new aldehyde oxidase selectively expressed in chemosensory organs of insects

Signal termination is a crucial step in the dynamic of the olfactory process. It involves different classes of odorant-degrading enzymes. Whereas aldehyde oxidase enzymatic activities have been demonstrated in insect antennae by previous biochemical studies, the corresponding enzymes have never been characterized at the molecular level. In the cabbage armyworm Mamestra brassicae, we isolated for the first time an aldehyde oxidase partial cDNA specifically expressed in chemosensory organs, with the strongest expression in antennae of both sexes. In these organs, expression was restricted to the olfactory sensilla. Our results suggest that the corresponding enzyme could degrade aldehyde odorant compounds, such as pheromones or plant's volatiles.     

Phenotypic biomonitoring using multivariate flow cytometric analysis of multi-stained microorganisms

A new method for monitoring phenotypic profiles of pure cultures and complex microbial communities was evaluated. The approach was to stain microorganisms with a battery of fluorescent dyes prior to flow cytometry analysis (FCM) and to analyse the data using multivariate methods, including principal component analysis and partial least squares. The FCM method was quantitatively evaluated using different mixtures of pure cultures as well as microbial communities. The results showed that the method could quantitatively and reproducibly resolve both populations and communities of microorganisms with 5% abundance in a diverse microbial background. The feasibility of monitoring complex microbial communities over time during the biodegradation of naphthalene using the FCM method was demonstrated. The biodegradation of naphthalene occurred to differing extents in microcosms representing three different types of aromatic-contaminated groundwater and a sample of bio-basin water. The FCM method distinguished each of these four microbial communities. The phenotypic profiles were compared with genotypic profiles generated by random-amplified polymorphic DNA analysis. The genotypic profiles of the microbial communities described only the microbial composition, and not their functional change, whereas the phenotypic profiles seemed to contain information on both the composition and the functional change of the microorganisms. Furthermore, event analysis of the FCM data showed that microbial communities with initially differing compositions could converge towards a similar composition if they had a capacity for high levels of degradation, whereas microbial communities with similar initial compositions could diverge if they differed in biodegrading ability.     

A novel method for determining rate constants for dehydration of aldehyde hydrates

A method has been developed for calculating rate constants for dehydration of aldehydes that induce ATPase reactions by kinases and where 18O is transferred from the aldehyde or its hydrate to inorganic phosphate during the reaction. The method involves measurement of the fraction of 18O in phosphate by 31P NMR after the ATPase reaction has proceeded for several minutes with zero-order kinetics. The reaction is started by addition of the aldehyde in a small volume of H2 18O, and the speed of washout of 18O by reversible dehydration relative to the rate of the ATPase reaction allows calculation of the rate constants if the hydration equilibrium constant is known from the proton NMR spectrum of the aldehyde. Dehydration rate constants (s-1 at pH 8-8.5, 0.1 M buffer, 25 degrees C) for the following aldehydes (all over 95% hydrated) and kinases used are as follows: D-glyceraldehyde with glycerokinase, 0.03; 2,5-anhydro-D-mannose 6-phosphate with fructose-6-phosphate kinase, 0.025; 2,5-anhydro-D-mannose or 2,5-anhydro-D-talose with fructokinase, 0.029 and 0.017, respectively; D-gluco-hexodialdose with hexokinase, 0.068. With betaine aldehyde and choline kinase or glyoxylate and pyruvate kinase, no 18O was transferred to phosphate during the ATPase reactions. However, the dehydration rate constant for glyoxylate (0.007 s-1 at pH 7 extrapolated to zero buffer concentration and up to 0.11 s-1 at pH 9.0 with 0.3 M buffer) was determined by extrapolating the initial rate of reduction of the free aldehyde catalyzed by lactate dehydrogenase to infinite enzyme levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.     

A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase

Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.     

Determination of kinetic constants for a two-stage anaerobic upflow packed-bed reactor for dairy wastewater

A two-stage upflow packed-bed (reactors in series) system was used for the treatment of dairy wastewater. Nylon pads were used as supporting media for the biomass. This investigation aimed at the determination of various kinetic constants for substrate, biomass and biogas based on various models. The maximum loadings that could be applied to reactor I and reactor II were 14·29 and 5·0 kg of chemical oxygen demand (COD) per m³ per day, respectively. The maximum COD removal efficiencies at various loading rates were in the ranges of 93·8–98·5% and 72·5–84% for the two reactor systems, respectively. The combined biogas yield was between 0·196 and 0·386 m³ gas/kg COD_a.     

A novel aldehyde dextran sulfonate matrix for affinity biosensors

Aldehyde dextran sulfonate (ADS), a modified oligosaccharide polymer, was used to prepare a new matrix structure for affinity biosensors. The principal difference between the ADS matrix and similar structures developed previously results from presence of two active functional groups in the matrix, namely, aldehyde and sulfonate. These groups perform two different functions in the matrix. The aldehyde group is responsible for covalent bonding in the biomaterials, and the negatively charged sulfonate group provides electrostatic attraction of the positively charged biomolecules. By varying the ratio between the aldehyde and sulfonate groups in the matrix, one can control contributions from the two binding modes (covalent and electrostatic). A number of oligosaccharides, such as simple dextran, aldehyde dextran (AD), aldehyde dextran sulfonate (ADS) and aldehyde ethylcellulose (AEC), were used for preparation of matrix structures. The properties of the obtained matrices were analysed and compared. Surface plasmon resonance (SPR) was used as the main technique to characterize the matrix structures.     

Steady-state kinetic mechanism of LodA,a novel cysteine tryptophylquinone-dependent oxidase

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of k_cat of 0.22 ± 0.04 s⁻¹, K_lysine of 3.2 ± 0.5 μM and K_O2 of 37.2 ± 6.1 μM. The k_cat exhibited a pH optimum at 7.5 while k_cat/K_lysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O₂ in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.     

Inhibition of guinea pig aldehyde oxidase activity by different flavonoid compounds: An in vitro study

Aldehyde oxidase (AO), a cytosolic molybdenum-containing hydroxylase, is predominantly active in liver and other tissues of mammalian species and involved in the metabolism of extensive range of aldehydes and nitrogen-containing compounds. A wide range of natural components including polyphenols are able to interfere with AO-catalyzed reactions. Polyphenols and flavonoids are one of the extensive secondary plant metabolites ubiquitously present in plants considered an important part of the human diet. The aim of the present study was to investigate inhibitory effect of selected phenolic compounds from three subclasses of aurone, flavanone and phenolic lactone compounds on the activity of AO, spectrophotometrically. AO enzyme was partially purified from liver of guinea pig. Then, inhibitory effects of 10 flavonoid compounds including 8 derivatives of 2-benzylidenebenzofuran-3(2H)-ones, as well as naringenin and ellagic acid on the activity of aldehyde oxidase were assessed compared with the specific inhibitor of AO, menadione. Among the phenolic compounds with inhibitory effects on the enzyme, ellagic acid (IC₅₀ = 14.47 μM) was the most potent agent with higher inhibitory action than menadione (IC₅₀ = 31.84 μM). The mechanisms by which flavonoid compounds inhibit AO activity have been also determined. The inhibitory process of the assessed compounds occurs via either a non-competitive or mixed mode. Although flavonoid compounds extensively present in the nature, mainly in dietary regimen, aurones with promising biological properties are not widely distributed in nature, so synthesis of aurone derivatives is of great importance. Additionally, aurones seem to provide a promising scaffold in medicinal chemistry for the skeleton of new developing drugs, so the results of the current study can be valuable in order to better understanding drug–food as well as drug–drug interaction and also appears to be worthwhile in drug development strategies.     

A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases

A simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k-1), acylation (k2), and deacylation (k3) in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+-binding and site-directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.     

Rapid screening of monoamine oxidase B inhibitors in natural extracts by capillary electrophoresis after enzymatic reaction at capillary inlet

A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N′-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH = 7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K_i, K_m and IC₅₀ based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD = 1.8%) and peak area (RSD = 3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.     

The determination of aldehyde oxidase activity patterns in the wing discs of Drosophila melanogaster

Summary The pattern of aldehyde oxidase (AO) activity was determined in wing discs of Drosophila melanogaster larvae homozygous for the mutants apt ⁷³ⁿ, Beaded, and vestigial (vg) in order to determine if reduction in field size in the pouch could be related to alterations of the wild-type AO pattern, as suggested by the Kauffman (1978) hypothesis. The pattern in wild-type discs was resolved into six areas for comparison with mutant discs. vg discs developed at 25° C showed restriction of the pattern into a small area on the anterior side of the disc, and comparison of vg and wild-type prepupal wings allowed positive identification of the AO pattern elements which remained. AO patterns in vg wing discs grown at 27°, 29°, and 31° C were progressively more complete and similar to wild-type, reflecting the reduction in cell death in discs grown at higher temperatures. These results show that cell loss during the third instar in vg development at 25° C is responsible for the alteration of the AO pattern, rather than field size reduction, and that determination of the pattern must take place much earlier than the time of its first appearance during the third larval instar, and before cell death in vg discs begins. Thus mutants acting at earlier stages will be necessary for further tests of the Kauffman hypothesis.     

A graphical method for determining inhibition constants

A new simple graphical method is described for the determination of inhibition type and inhibition constants of an enzyme reaction without any replot. The method consists of plotting experimental data as (V–v)/v versus the inhibitor concentration at two or more concentrations of substrate, where V and v represent the maximal velocity and the velocity in the absence and presence of inhibitor with given concentrations of the substrate, respectively. Competitive inhibition gives straight lines that converge on the abscissa at a point where [I]?=??K_i. Uncompetitive inhibition gives parallel lines with the slope of 1/K’_i. For mixed type inhibition, the intersection in the plot is given by [I]?=??K_i and (V–v)/v?=??K_i/K’_i in the third quadrant, and in the special case where K_i?=?K’_i (noncompetitive inhibition) the intersections occur at the point where [I]?=??K_i and (V–v)/v?=??1. The present method, the “quotient velocity plot,” provides a simple way of determining the inhibition constants of all types of inhibitors.     

Biosensing approach for alcohol determination using immobilized alcohol oxidase

Alcohol oxidase (AOD) was immobilized in polypyrrole (PPy) and a random copolymer containing 3-methylthienyl methacrylate and p-vinylbenzyloxy poly(ethyleneoxide) matrices. Immobilization of enzyme was performed via entrapment in conducting polymers during electrochemical polymerization of pyrrole through the thiophene moiety of the copolymer. Three different alcohols, namely methanol, ethanol and n-propanol, were used as substrates. Maximum reaction rates, Michaelis–Menten constants, optimum temperature and pH values, operational stabilities and shelf life of the enzyme electrodes were investigated.     

A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.     

Simultaneous determination of sucrose and trehalose in olive leaves by spectrophotometry utilizing partial least squares method

This study deals with the simultaneous determination of sucrose and trehalose in olive leaf samples, with easy sample preparation step, by second derivative UV–Vis spectrophotometry and partial least squares technique. A training set consisting of 31 binary mixture solutions was applied for the construction of PLS model. The proposed procedure was successfully applied for the simultaneous determination of both carbohydrates in laboratory prepared mixtures and in real samples. The real samples were from different growth stages of olive plant. The root mean square error of prediction was determined to be 1.95 and 2.06 for sucrose and trehalose, respectively. Also, limit of detection was 0.21 ppm for sucrose and 0.26 ppm for trehalose. The proposed procedure is rapid, simple, and easy to perform which can be used for routine analysis of both carbohydrates in plant leaves.     

A kinetic spectrophotometric method for simultaneous determination of glycine and lysine by artificial neural networks

A spectrophotometric method for simultaneous analysis of glycine and lysine is proposed by application of neural networks on the spectral kinetic data. The method is based on the reaction of glycine and lysine with 1,2-naphthoquinone-4-sulfonate (NQS) in slightly basic medium. On the basis of the difference in the rate between the two reactions, these two amino acids can be determined simultaneously in binary mixtures. Feed-forward neural networks have been trained to quantify considered amino acids in mixtures under optimum conditions. In this way, a one-layer network was trained. Sigmoidal and linear transfer functions were used in the hidden and output layers, respectively. Linear calibration graphs were obtained in the concentration range of 1 to 25microgml(-1) for glycine and 1 to 19microgml(-1) for lysine. The analytical performance of this method was characterized by the relative standard error. The proposed method was applied to the determination of considered amino acids in synthetic samples.     

A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase

An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively.