Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.11.1.7 (peroxidase)
 65,474 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C.V.) were <6.58% and <10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.
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PMID:Discrete analysis of serum uric acid with immobilized uricase and peroxidase. 1039 68

Urinary excretion of uric acid was found to be extremely low in a 58-year-old female patient with alcaptonuria. This was due to interference with the uricase-peroxidase method used, because analysis using high-performance liquid chromatography (HPLC) showed a normal urinary concentration of uric acid. In vitro experiments demonstrated that a high concentration of homogentisic acid in the patient's urine inhibited the peroxidase reaction, possibly due to inhibition of the colour development of 3-methyl-N-ethyl-N-(beta-hydroxyethyl)aniline (MEHA) and 4-aminoantipyrine, via the peroxidase reaction. A homogentisic acid concentration equivalent to that in plasma did not affect the uricase-peroxidase reaction. This result suggests that any assay based on a peroxidase method is affected by a high urinary concentration of homogentisic acid in patients with alcaptonuria.
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PMID:'Pseudohypouricosuria' in alcaptonuria: homogentisic acid interference in the measurement of urinary uric acid with the uricase-peroxidase reaction. 1045 13

A new enzyme-immobilization reaction by means of L-ascorbic acid (ASA) is described using NH(2) polymers based on cellulose or poly(vinyl alcohol) with the example of oxidoreductase enzymes. In this way, enzyme proteins such as glucose oxidase (GOD), glutamate oxidase, lactate oxidase, urate oxidase and peroxidase can be covalently fixed with a high surface loading to ultrathin and transparent NH(2)-polymer films if their surfaces are previously treated with an ASA solution, in, for example, N,N-dimethyl acetamide, DMSO or methanol. ASA then obviously reacts like a diketo compound with amino groups of the NH(2)-polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff's-base structures. In a subsequent fragmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH(2)-polymer film, as suggested by results from investigations of the ASA reaction with n-butylamine. The immobilized enzymes can be stored at 4 degrees C in bidistilled water for at least 1 month without becoming detached from the NH(2)-polymer film and without diminished enzyme activity. The apparent K(m) values of the immobilized enzymes are in part clearly smaller than those of the dissolved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the K(m) value of the immobilized GOD with different NH(2) polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.
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PMID:A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid. 1051 95

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.
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PMID:Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system. 1170 95

A chemiluminometric flow-through sensor for simultaneous determination of L-glutamate (Glu) and L-lysine (Lys) in a single sample has been developed. Immobilized uricase, immobilized peroxidase, support material, coimmobilized glutamate oxidase/peroxidase, support material, and coimmobilized lysine oxidase/peroxidase were packed sequentially in a transparent PTFE tube, and the tube was placed in front of a photomultiplier tube as a flow cell. A three-peak recording was obtained by one injection of the sample solution. The peak height of the first peak was due to the concentrations of urate and other reductants in the sample; the immobilized uricase was used to decompose urate, and the hydrogen peroxide produced was decomposed with a luminol-hydrogen peroxide reaction by immobilized peroxidase. The peak heights of the second and third peaks were free from the interferences from the reductants and were dependent only on the concentrations of Glu and Lys, respectively. Calibration graphs for Glu and Lys were linear at 40-1,000 and 50-1,200 nM, respectively. The sampling rate was 11/h without carryover. The sensor was stable for two weeks. The sensor system was applied to the simultaneous determination of Glu and Lys in serum.
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PMID:Chemiluminometric sensor for simultaneous determination of L-glutamate and L-lysine with immobilized oxidases in a flow injection system. 1192 93

A chemiluminescence biosensor on a chip coupled to a microfluidic system and a microreactor is described in this paper. The chemiluminescence biosensor measured 25 x 75 x 6.5 mm in dimension, and was readily produced in an analytical laboratory. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and luminol in the microreactor, and to immobilize uricase in the enzymatic reactor. The main characteristic of the biosensor was to introduce air as the carrier flow instead of the more common solution carrier for the first time. The uric acid was determined by a chemiluminescent (CL) reaction between the hydrogen peroxide produced from the enzymatic reactor and luminol under the catalysis of HRP in the microreactor. The linear range of the uric acid concentration was 1 to 100 mg L(-1) and the detection limit was 0.1 mg L(-1) (3sigma).
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PMID:Chemiluminescence biosensor chip based on a microreactor using carrier air flow for determination of uric acid in human serum. 1237 39

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.
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PMID:A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters. 1241 48

The simultaneous encapsulation of a coupled uricase-peroxidase system and amplex red in a sol-gel matrix allows one to obtain a reagent-less and ready-to-use fluorescent biosensor for the accurate detection of uric acid in highly diluted biological fluids. The detection limit of the prepared biosensor was found to be 20 nM and was linear up to 1 microM. The high sensitivity found for the biosensor permitted a reliable determination of uric acid concentrations in the presence of interfering species (e.g., ascorbic acid) just by sample dilution (up to 50000 for urine and 10000 for serum and blood). The sol-gel encapsulation preserved the hierarchy of the enzyme activity as demonstrated by the performance of the fluorescent biosensor.
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PMID:A reagent less fluorescent sol-gel biosensor for uric acid detection in biological fluids. 1459 33

This study describes a modified colorimetric assay for uricase activity in flexible 96-well microtiter plates using the uricase/uric acid/horseradish peroxidase/4-aminoantipyrine/3,5-dichloro-2-hydroxybenzene sulfonate colorimetric reaction. The utility of this assay was demonstrated in a screen for mutant uricase enzymes derived from the uricase gene of the thermophilic bacterium Bacillus subtilis by a modified staggered extension process (StEP) mutagenesis. An Escherichia coli library of StEP-derived uricase mutant clones was screened yielding two identical active mutant uricase genes. Two motifs conserved in eukaryotic and prokaryotic uricases are highly conserved in the mutant uricase. The mutant uricase protein was found to exhibit high uricase activity (13.1 U.mg(-1)). Finally, the modified colorimetric method is much more efficient than the conventional ones and greatly reduces assay time from 4 days to less than 20 h.
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PMID:Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis. 1472 78

An optical polymeric biochip system based on the complementary metal oxide semiconductor (CMOS) photo array sensor and polymeric enzyme biochip for rapidly quantitating uric acid in a one-step procedure was developed. The CMOS sensor was designed with N(+)/P-well structure and manufactured using a standard 0.5 microm CMOS process. The polymeric enzyme biochip was immobilized with uricase-peroxidase and used to fill the reacting medium with the sample. This study encompasses the cloning of the Bacillus subtilis uricase gene and expression in Escherichia coli, as well as the purification of uricase and measurement of its activity. The cloned uricase gene included an open reading frame of 1491 nucleotides that encodes a protein of approximately 55 kDa. The expression of the putative MBP-fusion protein involved approximately 98 kDa of the protein. The CMOS sensor response was stronger at a higher temperature range of 20-40 degrees C, with optimal pH at 8.5. The calibration curve of purified uric acid was linear in the concentration range from 2.5 to 12.5 mg/dL. The results obtained for serum uric acid correlated quite closely with those obtained using the Beckman Synchron method.
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PMID:Detection of serum uric acid using the optical polymeric enzyme biochip system. 1514 96


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