Xanthine oxidase assay

All the inhibitions reported so far indicate that there is no known specific inhibitor for the manganese superoxide dismutase. The enzyme from B. stearothermophilus was found to be inhibited by cacodylate (Thornalley et al., 1982) however, the inhibition could only be observed in the xanthine/xanthine oxidase assay and not in the pulse radiolysis assay. No evidence was obtained that cacodylate could be competing with the enzyme for because little or no inhibition was observed when the copper/zinc rather than the manganese enzyme was assayed in the presence of cacodylate. Further investigations revealed that the inhibitory effect is due to a cacodylate anion radical produced by the interaction of hydroxyl radicals (generated by the xanthine/xanthine oxidase reaction) and cacodylate anions. A radical of pamoic acid (4,4 -methylenebis-(3-... 

Significant antioxidant activity has also been observed in the hexane extracts of different cashew products (Table 10.3). The antioxidant activity was determined in the hypoxanthine/xanthine oxidase assay and expressed as inhibition of reactive oxygen species (ROS) attack on salicylic acid (%). CNSL has the highest antioxidant activity, followed by cashew fiber, cashew apple, raw cashew nut,... 

Antioxidant Activity of Hexane Extracts of Different Cashew Products as Determined in the Hypoxanthine/Xanthine Oxidase Assay... 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

K Withby, J Garson, A Baret. A microtiter format quantitative PCR assay for HCV RNA employing xanthine oxidase generated chemiluminescence. Proceedings of... 

Neither Suzuki et al. [206] nor Scott et al. [207] found any effect of LA on superoxide production by xanthine oxidase. Scott et al. also concluded that DHLA is incapable of reacting with superoxide. The last conclusion seems highly improbable. The ability of superoxide to react with thiols with the rate constants equal to 105 to 106lmol 1s 1 has been shown in chemical studies [208]. Dikalov et al. [209] estimated the rate constant for the reaction of DHLA with superoxide as (4.8 + 2)x 105 lmol-1 s-1 using the competition experiments with spin trap DMPO, which is very close to the previous value of (7.3+ 0.24) x 105 1 mol 1 s 1 reported for this reaction [210]. Negative results obtained by Scott et al. [207] are probably explained by the use of unreliable NBT assay for superoxide detection [211]. 

The basis of this assay was first used to measure the activity of superoxide dismutase (SOD) using a xanthine/xanthine oxidase 02"-generating system. O2 generated via this enzyme will reduce feni (oxidised)-cytochrome c, but SOD (which has a much higher affinity for O2" than cytochrome c) will prevent this reduction. Babior, Kipnes and Cumutte (1973) modified this technique to provide a specific assay to measure O2 production by activated neutrophils. Thus, 02" reduces cytochrome c (measured by an absorbance increase at 550 nm), but this reduction will be blocked by the addition of exogenous SOD (Fig. 5.10). 

In this indirect assay system, 05 is generated continuously by xanthine and xanthine oxidase, since 05 disproportionates spontaneously in the absence of a reactant for 05. In... 

The urinary caffeine test is not based on assays of specific substrates and products of NAT2 ( including other metabolism pathways involving at least xanthine-oxidases), and is affected by diet habits, xanthine-oxidase inhibitors such as allopurinol (Fuchs 1999), or other drugs (Klebovitch 1995). NAT activities are affected by anti-inflammatory drugs. Of note, acetominophen is an inhibitor of NAT2 in vivo (Rothen 1998). 

In Xanthine oxidase inhibition assay, the SFE extract show almost no bioactivity as the case of the LSE. In the oxygen free radical scavenger assay, the SFE extracts of Schizandrae fructus and Moutan Cortex Radicis were more active than the case of the LSE. 

A Oxygen radical scanvenger, Xanthine oxidase inhibition assay... 

Reaction conditions for hydroxylation of IPA included a phosphate buffer (pH 7.4) and the partially purified xanthine oxidase added to start the reaction. The reaction was stopped at 10-minute intervals with methanol, clarified by centrifugation, filtered, and analyzed by HPLC. The results of an assay are shown in Figure 9.116. 

Xanthine oxidase is a flavoenzyme that also contains molybdenum and iron. It shows activity toward a variety of substrates including purines, pyrimidines, aldehydes, and NADH. In this assay, isoxanthopterin is produced from 2-amino-4-hydroxypteridine. 

Luminescent endpoints are available for all of the commonly used enzyme labels, including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, glucose oxidase, and P-galactosidase. Detection limits in the subattomolar range have been attained (34). Chemiluminescent assays for novel enzyme labels such as xanthine oxidase have also been developed (50). 

The salvage pathway does not involve the formation of new heterocyclic bases but permits variation according to demand of the state of the base (B), i.e. whether at the nucleoside (N), or nucleoside mono- (NMP), di- (NDP) or tri- (NTP) phosphate level. The major enzymes and routes available (Scheme 158) all operate with either ribose or 2-deoxyribose derivatives except for the phosphoribosyl transferases. Several enzymes involved in the biosynthesis of purine nucleotides or in interconversion reactions, e.g. adenosine deaminase, have been assayed using a method which is based on the formation of hydrogen peroxide with xanthine oxidase as a coupling enzyme (81CPB426). 

Enzyme assays Cytidine deaminase xanthine oxidase... 

Most stilbenoids possess antioxidant activities because they possess polyphenol functions in the molecules. Some of their beneficial effects, hepatoprotective action, cardiovascular protection, for instance, are in close relation to their antioxidant activities. Several models have been employed in the assay such as lipid peroxidation system, human low-density lipoprotein model, xanthine oxidase system and l,l-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging model, which is the most commonly used protocol. 

In a commercially available assay, serum NTP catalyzes the hydrolysis of IMP to yield inosine, which is then converted to hypoxanthine by purine-nucleoside phosphorylase (EC 2.4.2.1). Hypoxanthine is oxidized to urate with xanthine oxidase (EC 1.2.3.2). Two moles of hydrogen peroxide are produced for each mole of hypoxanthine liberated and converted to uric acid. The formation rate of hydrogen peroxide is monitored by a spectrophotometer at 510nm by the oxidation of a chromogenic system. The effect of ALPs on IMP is inhibited by p-glycerophosphate. This material is substrate for ALP but not for NTP, and by forming substrate complexes with the former enzyme, it reduces the proportion of the total ALP activity that is directed to the hydrolysis of the NTP substrate, IMP. ... 

Watanabe et al. (1986) developed a sequence sensor for the successive assay of hypoxanthine (HX) and inosine (HXR) by arranging nucleoside phosphorylase (EC 2.4.2.1) and xanthine oxidase (EC 1.2.3.2) in spatially separated layers in front of an oxygen probe. Nucleoside phosphorylase was... 

Since inorganic phosphate is indispensable for the nucleoside phosphorylase reaction, the phosphate concentration can be converted into an oxygen signal by using a nucleoside phosphorylase-xanthine oxidase sequence electrode (Watanabe et al., 1987 Watanabe, 1988). 2 mmol/1 of inosine has been shown to be necessary for optimal sensitivity to phosphate ion. The measuring range was 0.1-1 mmol/1 and the sensor could be used for 70 assays. 

Because the enzyme substrate and the products are unstable, it is difficult to measure the disappearance of substrate or the formation of products as is usual in enzymatic assays. Routine assays for SOD usually employ an indirect assay in which one unit of enzyme activity is defined as the amount of enzyme that inhibits the reaction of 02 with the indicator by 50%. The most frequently used method for measuring SOD activity employs the xanthine/xanthine oxidase reaction for... 

In the xanthine/xanthine oxidase-cytochrome c method originally developed by McCord and Fridovich (M19), a typical assay mixture consists of oxidized1 cytochrome c, xanthine, sufficient xanthine oxidase, and phosphate buffer at pH 7.8 containing EDTA in a total volume of 3 ml. The rate of reaction is followed at 550 nm. One unit of SOD activity is defined as the amount that causes 50% inhibition of the rate of reduction of cytochrome c. 

Fig. 27 (79). Reduction of oxidized cytochrome c by Og- produced during the enzy-mic-catalysed oxidation of xanthine. in the presence of catalase (16 nM), erythrocuprein omitted after addition of erythrocuprein (25 nM) and catalase (16 nM) without either erythrocuprein or catalase in the presence of erythrocuprein (25 nM), catalase omitted in the presence of both catalase (16 nM) and p-chloro-mercuri-benzoic acid (400 /iM). The assay was performed at 25 °C in a volume of 0.76 ml. The concentration of the different components dissolved in 50 mM phosphate buffer, pH 7.8 was Xanthine 0.33 mM cytochrome cox, 27 / M EDTA, 0.1 mM xanthine oxidase, 0.21 fiM. The reduction of cytochrome c was recorded in a Unicam SP 1800 spectrophotometer at 550 nm...

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