Glucose-6-phosphate dehydrogenase 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). 

Figure 7-10. Coupled enzyme assay for hexokinase activity. The production of glucose 6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP". When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose 6-phosphate by hexokinase.

Another common procedure which is used for glucose assay is the hexokinase procedure in which the glucose is phosphory-lated by means of ATP and then dehydrogenated with glucose-6-phosphate dehydrogenase measuring the abosrption of NADPH... 

Bishop, C. Assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in red cell. J. 

W. Huang, A. Feltus, A. Witkowski, and S. Daunert, Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase-bacterial luciferase enzyme system. Anal. Chem. 68, 1646-1650 (1996). 

El. Ells, H. A., and Kirkman, H. N., A colorimetric method for assay of erythrocytic glucose-6-phosphate dehydrogenase. Froc. Soc. Exptl. Biol. Med. 106, 607-609 (1961). 

G10. Glock, G. E., and McLean, P., Further studies on the properties and assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. ]. 55, 400-408 (1953). 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

Very low concentrations of substrates may be assayed by recycling the test substrate for an appreciable but definite period of time and measuring the amount of product formed. The coenzyme NADPH, for instance, may be assayed using the two enzymes glutamate dehydrogenase (EC 1.4.1.3) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) ... 

Glucose-6-phosphate dehydrogenase is used as an indicator enzyme in the hexokinase assay of glucose... 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

The intermediate, NAD- or NADP-, is a radical on the nicotinamide that can react with [(bpy)3Ru]3+. Any enzyme that produces or consumes either NADH or NADPH can be directly monitored by ECL since only the reduced forms NAD(P)H but not the oxidized forms NAD(P)+ can function as a coreactant [31,49], This difference has been exploited in the clinical chemistry assays of ethanol, glucose, bicarbonate, cholesterol, and glucose-6-phosphate dehydrogenase. 

Protein concentrations are less than 0.1 mg/mL in two cases to avoid over-metabolism of the substrate. The buffer consists of potassium phosphate (50 mM, pH 7.4), MgCl2 (3 mM), EDTA (1 mM), and the NADPH-generating system [NADP (1 mM), glucose-6-phosphate (5 mM), and glucose-6-phosphate dehydrogenase (1 U/mL)]. The incubation time is five minutes for all assays. 

The first method of enzymatic PolyP assay was proposed by Clark et al. (1986). In this technique, PolyPs were determined by polyphosphate glucokinase obtained from Pro-pionibacterium shermanii. Glucose-6-phosphate dehydrogenase reduced NADP through utilization of the formed glucose-6-phosphate, and the increase in NADPH concentration was measured. 

Figure 15-3 Coupled enzyme assay to determine glycogen phosphorylase activity. As long as the activity of phosphoglucomutase and glucose-6-phosphate dehydrogenase are not rate-limiting, the AA340/At is directly proportional to the A[glucose-l -phosphate]/Atime.

Dispense 2.7-ml of this reaction cocktail in 10 small glass test tubes (13 X 100 mm), labeled A through J. To each of the 10 test tubes, add 100 (A of the 10-units/ml glucose-6-phosphate dehydrogenase solution and 100 /A of the 10-units/ml phosphoglucomutase solution. The total volume in each test tube after this step will be 2.9 ml. Refer to Table 15-2 to aid you in setting up the rest of the assay tubes. 

Total phosphorus is determined as inorganic phosphate after treatment with hot perchloric acid. Phosphorus in D-glucose-6-phosphate residues is assayed using D-glucose-6-phosphate dehydrogenase. 

The reaction mixture contained Tris-HQ buffer (pH 7.4), glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and the microsomes. The mixture was preincubated, and the reaction was started by the addition of BaP dissolved in acetone. The mixture was incubated for 30 minutes at 37°C and terminated by the addition of cold acetone. Samples were diluted and injected for analysis. An example of the assay results is shown in Figure 9.126. The activity was from rat microsomes. 

The assay mixture contained imidazole (pH 7.2), KC1, NADPH, glucose-6-phosphate, DTT, glucose-6-phosphate dehydrogenase, and H2-biopterin. Samples were incubated for 60 minutes with tissue extract and terminated with 2 N TCA. After centrifugation (15,000g for 5 min), the H4-biopterin was oxidized to pterin with iodine, the reaction was terminated with ascorbic acid, and samples were injected onto the HPLC column for analysis. An example of an assay is shown in Figure 9.131, and a composite of the rate of product formation is shown in Figure 9.132. 

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). 

At 25, the assays contained 0.2 mAf DTNB and the appearance of TNB anion was followed at 412 nm the assays also contained 10 mAf glucose 6-phosphate and glucose-6-phosphate dehydrogenase to overcome inhibition by NADP/ except in the experiment estimating NADP inhibition. 

Enzymatic reactions that cannot be monitored directly by spectroscopic changes can be coupled to other reactions that do show such changes. One of the classic examples is the detection of glucose by an assay which depends on its conversion by hexo-kinase into glucose 6-phosphate, which is then coupled to an ancillary indicator reaction with NADP+ and glucose 6-phosphate dehydrogenase ... 

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