A sensitive spectrophotometric assay for guanase activity

A highly sensitive and accurate spectrophotometric method was developed for determination of guanase activity with guanine as substrate. The assay is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone and N,N-diethylaniline. Xanthine formed from guanine by guanase is oxidized to uric acid and hydrogen peroxide by xanthine oxidase, and the hydrogen peroxide produced is determined by an oxidative-coupling reaction with 3-methyl-2-benzothiazolinone hydrazone and N,N-diethylaniline mediated by peroxidase. Formation of the indamine dye is greatly affected by the superoxide radical ion (O2-) and pH value. These problems can be overcome by separating the two reactions of hydrogen peroxide formation and color production and carrying out that color-producing reaction at pH 3.0. This method is very sensitive and accurate because the indamine dye has a very high molar extinction coefficient of 29,800. It can be used with various kinds of automatic analyzers such as a Hitachi, Olympus, or Technicon analyzer. Comparative studies showed that this method is more sensitive and reproducible than other methods. Furthermore, guanase activities determined by this method correlated well with those determined by the improved Ellis-Goldberg method. This method should be useful for measurement of guanase activity in banked blood for preventing transfusion hepatitis and could be valuable as a liver function test.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Enhancement by nitrite of peroxide-induced degradation of uric acid and 3-N-ribosyluric acid

The oxidation of uric acid and 3-N-ribosyluric acid by hydrogen peroxide and methemoglobin was stimulated by the addition of sodium nitrite, which alone has no effect on the urates. The urates were not oxidized by either hydrogen peroxide alone or hydrogen peroxide and sodium nitrite unless methemoglobin was present. t-Butyl hydroperoxide also oxidized the urates in the presence of methemoglobin, but the reaction was not stimulated by sodium nitrite. The addition of either sodium azide or potassium cyanide reduced the rate of the reaction with either hydrogen peroxide or t-butyl hydroperoxide both in the presence and absence of sodium nitrite. Possible explanations for the stimulation by nitrite of peroxide-induced degradation of urates are presented.     

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.     

A new spectrophotometric assay for enzymes of purine metabolism. I. Determination of xanthine oxidase activity.

A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.     

Free radical metabolite of uric acid

Uric acid has previously been shown to act as a water-soluble antioxidant. Although the antioxidant activity of uric acid has been attributed to its ability to scavenge free radicals, the one-electron uric acid oxidation product of such a scavenging reaction has not been detected. It order to determine whether a free radical metabolite of uric acid could be formed via one-electron redox processes, we oxidized uric acid with potassium permanganate, horseradish peroxidase/hydrogen peroxide, and hematin/hydrogen peroxide systems. With the use of the rapid-mixing, continuous-flow electron spin resonance technique, we were able to detect the urate anion free radical in all three radical-generating systems. Based on N15-isotopic-labeling experiments, we show that the unpaired electron of this radical is located primarily on the five-membered ring of the purine structure. We were also able to demonstrate that this radical could be scavenged by ascorbic acid.     

The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla.

The formation of hydrogen peroxide during the oxidation of NADH by purified preparations of cytochrome o has been demonstrated by employing three independent methods: polarographic, colorimetric, and fluorometric. The first two methods were used to assay for the accumulation of hydrogen peroxide and showed that hydrogen peroxide did accumulate as a product, but only about 30% of the oxygen consumed or 15 to 20% of the NADH oxidized was recoverable as hydrogen peroxide. This lack of 1:1 stoichiometry was not due to residual catalase activity in these preparations which could be eliminated by freeze-thawing. Thus, hydrogen peroxide may not be the sole or primary product of the NADH-cytochrome o oxidase reaction. The fluorometric assay could be coupled directly to the NADH-cytochrome o oxidase reaction in one medium, and this method showed that hydrogen peroxide was generated continuously from the beginning of the reaction in a 1:1 stoichiometry, hydrogen peroxide generated to NADH oxidized. This result suggests that hydrogen peroxide is an intermediate that can be trapped efficiently under the conditions of the fluorometric assay, whereas under the conditions of the first two assays most of the hydrogen peroxide generated undergoes further reaction. Exogenously added FAD or FMN increased the percentage of hydrogen peroxide that accumulated in the NADHcytochrome o oxidase reaction. Flavin is believed to act on the reductase side of cytochrome o so the increased percentage of hydrogen peroxide is not likely to result from the direct reaction of reduced flavin with oxygen.     

Catalase deficiency may complicate urate oxidase (rasburicase) therapy

Patients with low (inherited and acquired) catalase activities who are treated with infusion of uric acid oxidase because they are at risk of tumour lysis syndrome may experience very high concentrations of hydrogen peroxide. They may suffer from methemoglobinaemia and haemolytic anaemia which may be attributed either to deficiency of glucose-6-phosphate dehydrogenase or to other unknown circumstances. Data have not been reported from catalase deficient patients who were treated with uric acid oxidase. It may be hypothesized that their decreased blood catalase could lead to the increased concentration of hydrogen peroxide which may cause haemolysis and formation of methemoglobin. Blood catalase activity should be measured for patients at risk of tumour lysis syndrome prior to uric acid oxidase treatment.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

Catalase deficiency may complicate urate oxidase (rasburicase) therapy

Patients with low (inherited and acquired) catalase activities who are treated with infusion of uric acid oxidase because they are at risk of tumour lysis syndrome may experience very high concentrations of hydrogen peroxide. They may suffer from methemoglobinaemia and haemolytic anaemia which may be attributed either to deficiency of glucose-6-phosphate dehydrogenase or to other unknown circumstances. Data have not been reported from catalase deficient patients who were treated with uric acid oxidase. It may be hypothesized that their decreased blood catalase could lead to the increased concentration of hydrogen peroxide which may cause haemolysis and formation of methemoglobin. Blood catalase activity should be measured for patients at risk of tumour lysis syndrome prior to uric acid oxidase treatment.     

Interference elimination of an amperometric glucose biosensor using poly(hydroxyethyl methacrylate) membrane

A permselective membrane fabricated from photo‐cross‐linked poly(hydroxyethyl methacrylate) (pHEMA) was studied as a potential selective membrane that can eliminate electrochemical interferences commonly faced by a hydrogen peroxide‐based biosensor. The quantitative selection of the permselective membrane was based on the permeabilities of hydrogen peroxide and acetaminophen (AC). AC was used as a model of the interfering substance due to its neutral nature. pHEMA membrane with the cross‐linking ratio of 0.043 was found to achieve a selectivity of hydrogen peroxide over AC of 10, while maintaining an acceptable degree of hydrogen peroxide response. A two‐layer glucose biosensor model consisting of glucose oxidase entrapped within a freeze‐thawed poly(vinyl alcohol) matrix and the cross‐linked pHEMA membrane was challenged with AC, ascorbic acid and uric acid. 0.2 mM AC and 0.2 mM ascorbic acid were completely eliminated. However, 0.2 mM uric acid could not be completely eliminated and still gave a bias of approximately 6.6% relative to 5 mM glucose. The results showed that cross‐linked pHEMA was quite promising as an interference eliminating inner membrane.     

X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase

Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid using gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide in absence of any cofactor or transition metal. The catalytic mechanism was investigated using X-ray diffraction, electron spin resonance spectroscopy (ESR), and quantum mechanics calculations. The X-ray structure of the anaerobic enzyme-substrate complex gives credit to substrate activation before the dioxygen fixation in the peroxo hole, where incoming and outgoing reagents (dioxygen, water, and hydrogen peroxide molecules) are handled. ESR spectroscopy establishes the initial monoelectron activation of the substrate without the participation of dioxygen. In addition, both X-ray structure and quantum mechanic calculations promote a conserved base oxidative system as the main structural features in UOX that protonates/deprotonates and activate the substrate into the doublet state now able to satisfy the Wigner's spin selection rule for reaction with molecular oxygen in its triplet ground state.     

In situ heterogeneity of peroxisomal oxidase activities: An update

Summary Oxidases are a widespread group of enzymes. They are present in numerous organisms and organs and in various tissues, cells, and subcellular compartments, such as mitochondria. An important source of oxidases, which is investigated and discussed in this study, are the (micro)peroxisomes. Oxidases share the ability to reduce molecular oxygen during oxidation of their substrate, yielding an oxidized product and hydrogen peroxide. Besides the hydrogen peroxide-catabolizing enzyme catalase, peroxisomes contain one or more hydrogen peroxide-generating oxidases, which participate in different metabolic pathways. During the last four decades, various methods have been developed and elaborated for the histochemical localization of the activities of these oxidases. These methods are based either on the reduction of soluble electron acceptors by oxidase activity or on the capture of hydrogen peroxide. Both methods yield a coloured and/or electron dense precipitate. The most reliable technique in peroxisomal oxidase histochemistry is the cerium salt capture method. This method is based on the direct capture of hydrogen peroxide by cerium ions to form a fine crystalline, insoluble, electron dense reaction product, cerium perhydroxide, which can be visualized for light microscopy with diaminobenzidine. With the use of this technique, it became clear that oxidase activities not only vary between different organisms, organs, and tissues, but that heterogeneity also exists between different cells and within cells, i.e. between individual peroxisomes. A literature review, and recent studies performed in our laboratory, show that peroxisomes are highly differentiated organelles with respect to the presence of active enzymes. This study gives an overview of thein situ distribution and heterogeneity of peroxisomal enzyme activities as detected by histochemical assays of the activities of catalase, and the peroxisomal oxidasesd-amino acid oxidase,l--hydroxy acid oxidase, polyamine oxidase and uric acid oxidase.     

The mechanism of peroxidase-mediated cytotoxicity. II. Role of the heme moiety

Various peroxidases in the presence of hydrogen peroxide and a halide ion have been shown to exert a cytolytic activity against erythrocytes and other cells. However, few studies have been done to elucidate the active site on the enzymes that is responsible for the cytotoxic activity. In addressing this question we found that boiling of horseradish peroxidase only partially abolishes its cytotoxic activity, suggesting that an intact tertiary structure of the protein may not be essential for the cytotoxic activity. This conclusion was confirmed by demonstrating that microperoxidase, hemin, and hematoheme also exert cytotoxic activity in the presence of hydrogen peroxide and iodide, the kinetics of which were identical to those obtained with the peroxidases. Fluoride, bromide, and thiocyanate could not replace iodide in any of these systems. These results indicate that the active site for the cytotoxic activity of the peroxidases is located within the heme moiety, whereas the protein portions of the enzymes affect the cytotoxic activity of the enzymes only in an indirect manner. We also tested a variety of compounds for their ability to inhibit the cytolytic reaction toward erythrocytes. We found that compounds such as thiourea, thionicotinamide, and uric acid are much more potent inhibitors of the cytolytic reaction than tyrosine and histidine. These observations support the concept that oxidative reactions rather than halogenation reactions are the primary cause of the peroxidase-mediated lysis of erythrocytes.     

The oxidation and decarboxylation of retinoic acid by horseradish peroxidase.

The decarboxylation of retinoic acid by horseradish peroxidase was investigated. A marked increase in the yield of products was obtained. However, the data indicated the reaction was a nonenzymatic, heme catalyzed peroxidation. Previously reported requirements for phosphate, oxygen and ferrous ion were eliminated when hydrogen peroxide was provided. Peroxide also eliminated the EDTA and cyanide induced inhibition of the phosphate dependent system. In the presence of hydrogen peroxide, horseradish peroxidase was not essential to the reaction; heme equivalent amounts of hemoglobin decarboxylated retinoic acid with equal facility. However, hemoglobin was ineffective in the absence of hydrogen peroxide. Attainment of 50--60% decarboxylation represented complete utilization of the available retinoic acid. Thus the products of the reaction can be divided into two groups, products of retinoic acid oxidation and products of an oxidative decarboxylation of retinoic acid.     

Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen.

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.     

A nonenzymatic method for determination of hydrogen peroxide and organic peroxides

Reduction of hydrogen peroxide and organic peroxides (t-butyl hydroperoxide and linoleic acid hydroperoxide) was achieved with homovanillic acid as hydrogen donor in the presence of the triethylenetetramine-Fe3+ complex. By the catalytic action of this complex, homovanillic acid is oxidized to its fluorescent dimer. Based on this reaction a fluorometric method for the measurement of the hydroperoxides mentioned above is described. The method can be extended to the determination of substrate-enzyme systems that produce hydrogen peroxide, e.g., glucose-glucose oxidase. The method allows the determination of substances such as hydrogen peroxide and t-butyl hydroperoxide with an accuracy and precision of less than 3%. Glucose can be determined with similar precision and an accuracy of 4.7%.     

Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial fraction

1. The primary intermediate of catalase and hydrogen peroxide was identified and investigated in peroxisome-rich mitochondrial fractions of rat liver. On the basis of kinetic constants determined in vitro, it is possible to calculate with reasonable precision the molecular statistics of catalase action in the peroxisomes. 2. The endogenous hydrogen peroxide generation is adequate to sustain a concentration of the catalase intermediate (p(m)/e) of 60-70% of the hydrogen peroxide saturation value. Total amount of catalase corresponds to 0.12-0.15nmol of haem iron/mg of protein. In State 1 the rate of hydrogen peroxide generation corresponds to 0.9nmol/min per mg of protein or 5% of the mitochondrial respiratory rate in State 4. 3. Partial saturation of the catalase intermediate with hydrogen peroxide (p(m)/e) in the mitochondrial fraction suggests its significant peroxidatic activity towards its endogenous hydrogen donor. A variation of this value (p(m)/e) from 0.3 in State 4 to 0 under anaerobic conditions is observed. 4. For a particular preparation the hydrogen peroxide generation rate in the substrate-supplemented State 4 corresponds to 0.17s(-1) (eqn. 6), the hydrogen peroxide concentration to 2.5nm and the hydrogen-donor concentration (in terms of ethanol) to 0.12mm. The reaction is 70% peroxidatic and 30% catalatic. 5. A co-ordinated production of both oxidizing and reducing substrates for catalase in the mitochondrial fraction is suggested by a 2.2-fold increase of hydrogen peroxide generation and a threefold increase in hydrogen-donor generation in the State 1 to State 4 transition. 6. Additional hydrogen peroxide generation provided by the urate oxidase system of peroxisomes (8-12nmol of uric acid oxidized/min per mg of protein) permits saturation of the catalase with hydrogen peroxide to haem occupancy of 40% compared with values of 36% for a purified rat liver catalase ofk(1)=1.7x10(7)m(-1).s(-1) and k'(4)=2.6x10(7)m(-1). s(-1)(Chance, Greenstein & Roughton, 1952). 7. The turnover of the catalase ethyl hydrogen peroxide intermediate (k'(3)) in the peroxisomes is initially very rapid since endogenous hydrogen peroxide acts as a hydrogen donor. k'(3) decreases fivefold in the uncoupled state of the mitochondria.     

A micromachined capillary electrophoresis chip with fully integrated electrodes for separation and electrochemical detection

A novel analytical microsystem with fully integrated electrodes for electrophoresis and amperometrical detection is described. With respect to the lab-on-a-chip concept a capillary electrophoresis (CE) microsystem has been fabricated with a total of six gold electrodes for sample injection, separation and electrochemical detection using standard microfabrication technologies. The device is a ready-to-use system that does not need any extra mechanical apparatus for electrode insertion. The CE-chip has successfully been tested by measuring hydrogen peroxide, ascorbic acid and uric acid simultaneously. All three oxidizable species could be detected in less than 70 s. Glucose was detected by performing an enzymatic reaction along the separation channel. The microsystem showed a very good reproducibility.     

Reaction of methylated urates with 1,1-diphenyl-2-picrylhydrazyl

The kinetics of the reaction between the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and methylated urates was studied. Urates that had methyl groups on the 1,3,9, or on the 1 and 3 or 1 and 9 nitrogens reacted with DPPH 15 to 77% faster than uric acid. Urates substituted with methyl groups on the 7 nitrogen or on both the 3 and 9 nitrogens reacted with DPPH at rates that were less than 0.1 that of uric acid. 3,7,9-Trimethyluric acid and 1,3,7,9-tetramethyluric acid reacted with DPPH at barely detectable rates. DPPH reacted with uric acid, the monomethylated urates, and some of the dimethylated urates in a ratio of 2:1. DPPH reacted with other dimethylated and trimethylated urates in a ratio of 1:1. Semiempirical MNDO calculations indicate that the most stable radical of uric acid is formed by hydrogen abstraction from the 3, 7 or 9 position. The most stable species resulting from loss of a second hydrogen lack hydrogens at the 3 and 7 positions or the 7 and 9 positions. For maximum reactivity with DPPH, methylated uric acid derivatives must have a hydrogen at nitrogen 7 and one of the hydrogens at either the 3 or 9 position.