Hydrogen peroxide-generating oxidases determination

VIII. DETERMINATION OF HYDROGEN PEROXIDE-GENERATING OXIDASES... 

A fiberoptic biosensor has been used for the determination of xanthine and hypoxanthine by immobilization of xanthine oxidase and peroxidase on different preactivated membranes, which were mounted onto the tip of the fiberoptic bundle [47], The hydrogen peroxide generated was measured using the luminol reaction. A linear calibration curve of the sensors occurred in the range of 1-316 nM hypoxanthine and of 3.1-316 nM xanthine, respectively, with a detection limit of 0.55 nM. 

The same Swedish group has designed a FIA system containing even more packed reactors and, indeed, a dialysis unit [758]. Aimed at the determination for galactose, the manifold for this system is shown in Fig. 4.50. Again, the final measurement is based on determination of hydrogen peroxide generated by an enzymatic reaction of an oxidase ... 

Reversed-phase h.p.l.c. with appropriate on-line immobilized oxidase enzyme reactors and an electrochemical detector to determine the hydrogen peroxide generated, proved sensitive (nM to pM detection limits), selective and rapid, and was applicable to the analysis of sugars in foodstuffs or... 

On the other hand, several oxidases are known to generate hydrogen peroxide, acting as an oxidant in the CL system, from corresponding substrates. IMERs in which the oxidases are immobilized on adequate supporting materials such as glass beads have been developed. IMERs are often used for flow injection with CL detection of uric acid and glucose, and are also applicable to the CL determination of acetylcholine, choline, polyamines, enzyme substrates, etc., after online HPLC separation. 

ALTERNATE PROTOCOL 2 ENZYMATIC MEASUREMENT OF CHOLESTEROL Test combination kits for enzymatic determination of cholesterol in food are now commercially available. For the determination of total cholesterol, esterified cholesterol is hydrolyzed to free cholesterol and fatty acid under mild alkaline conditions. Cholesterol oxidase oxidizes free cholesterol to cholest-4-en-3-one to generate hydrogen peroxide, which further oxidizes methanol to formaldehyde. Formaldehyde then reacts with acetyl acetone in the presence of NH4+ ions to form yellow lutidine dye, which is subsequently determined spectrophotometric al 1 y. 

There are many oxidases used in clinical and analytical chemistry for the specific determination of constituents of complex samples, based on the generation of hydrogen peroxide by the action of an aerobic oxidase on its substrate. Cholesterol, glucose, galactose and many other analytes are determined that way. These analyses are also important in food analysis. But the volume of oxidases consumed in food processing is primarily based on the protection afforded the food by the removal of either oxygen or the other substrate acted upon. 

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