Cholesterol oxidase oxidation

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. 

Figure C1.5.17.(A) Enzymatic cycle of cholesterol oxidase, which catalyses tire oxidation of cholesterol by molecular oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate,...

A new cholesterol flow injection analysis biosensor has also been described as an application of the H2O2 ECL sensor56. In that work, the luminol electrochemiluminescence, previously studied in aqueous media, was implemented in Veronal buffer added of 0.3% triton X-100 (v/v), 0.3% PEG and 0.4% cholate to enable the solubilisation of the cholesterol and then its efficient oxidation catalyzed by the immobilized cholesterol oxidase. The ECL reaction occurred thus in a micellar medium and the performances of the H2O2 ECL sensor were investigated. 

The enzyme cholesterol oxidase is an effective oxidation catalyst when used in a micellular system involving scCC>2 and water [25], The use of a perfluoropoly-ether-based surfactant causes the formation of reverse-micelles in which the... 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

Griffiths et al. [23] studied the ESI MS of sterols, and while they are not hormonal steroids, similar derivatization methods can be used. He converts 3/5-hydroxy-A5 sterols to - -4-ene sterols using cholesterol oxidase and follows this by preparation of Girard P hydrazones. This increases the sensitivity by 1000 in ESI. This technique would also be applicable to pregnenolone, dehydroepiandrosterone (DHEA) and similar A5 steroids, which can also be oxidized by cholesterol oxidase. 

Randolph et al. (1988) Batch/semibatch Oxidation of cholesterol Cholesterol oxidases Geioeocysticum chrysocreas and Streptomyces sp. 

Enzyme catalyzed oxidations with 02 have also been successfully performed in scC02 e.g. using cholesterol oxidase [95] and polyphenol oxidase [88]. The use of scC02 as a solvent for biotransformations clearly has considerable potential and we expect that it will find more applications in the future. 

In work published in 1989, Hylemon et al. used cholesterol oxidase to convert 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one. Cholesterol that remained was converted to 4-cholesten-3-one. 7/3-Cholesterol, which was added as an internal steroid recovery standard, was oxidized to 7/3-hydroxy-4-cholesten-3-one. These steroid products were analyzed by Qg reversed-phase chromatography on an Altex Ultrasil-ODS column (4.6 mm x 25 cm) using 70 30 (v/v) mixture of acetonitrile and methanol (Fig. 9.83). The eluate was monitored at 240 nm, and the amount of product determined from a calibration curve. 

Sensors have also been constructed from some oxidases directly contacted to electrodes to give bioelectrocatalytic systems. These enzymes utilize molecular oxygen as the electron acceptor for the oxidation of their substrates. Enzymes such as catechol oxidase, amino acid oxidase, glucose oxidase, lactate oxidase, pyruvate oxidase, alcohol oxidase, xanthine oxidase and cholesterol oxidase catalyze the oxidation of their respective substrates with the concomitant reduction of O2 to H2O2 ... 

In 1998, we reported the real-time observation of enzymatic turnovers of a single-molecule cholesterol oxidase, a flavoenz3mie that catalyzes oxidation of cholesterol by oxygen [15] (Fig. 22.lA). The active site of the enz3une, flavin adenine dinucleotide (FAD), (Fig. 22.IB), is naturally fluorescent in its oxidized form but not in its reduced form. With excess amounts of cholesterol... 

Fig. 22.1. (A) Enzymatic cycle of cholesterol oxidase which catalyzes the oxidation of cholesterol by oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate molecule, generating a non-fluorescent FADH2, which is then oxidized by oxygen. (B) Structure of FAD, the active site of cholesterol oxidase. (C) A portion of the fluorescence intensity time trace of a single cholesterol oxidase molecule. Each on-off cycle of emission corresponds to an enzymatic turnover. (D) Distribution of emission on-times derived from (C). The solid line is the convolution of two exponential functions with rate constants fci[S] = 2.5 s and fc2 = 15.3 s, reflecting the existence of an intermediate, ES, the enzyme-substrate complex, as shown in the kinetic scheme in the inset. From ref. [15]...

Similar optical biosensors have been prepared for many other analytes. For example, a cholesterol optical biosensor has been devised based on fluorescence quenching of an oxygen-sensitive dye that is coupled to consumption of oxygen resulting from the enzyme-catalyzed oxidation of cholesterol by the enzyme cholesterol oxidase. Serum bilirubin has been detected using bilirubin oxidase, coimmobilized with a ruthenium dye, on an optical fiber.The bilirubin sensor was reported to exhibit a lower detection limit of iO Xmol/L, a linear range up to 30mmol/L, and a typical reproducibihty of 3% (CV), certainly adequate for clinical application. 

The 3-OH group of cholesterol is then oxidized to a ketone in an oxygen-requiring reaction catalyzed by cholesterol oxidase,... 

Khmelnitsky, Y. L., HUhorst, R., and Veeger, C., Detergentless microemulsions as a media for enzymatic reactions cholesterol oxidation catalyzed by cholesterol oxidase, Eur. J. Biochem., 176, 265-271, 1988. 

The basis for all enzymatic cholesterol assays is the hydrolysis of cholesterol esters by cholesterol esterase (CEH, EC 3.1.1.13) to free cholesterol and fatty acids and the oxidation of free cholesterol to cholestenone by cholesterol oxidase (COD, EC 1.1.3.6) with concomitant oxygen consumption and hydrogen peroxide formation ... 

Fig. 63. Dependences of the hydrogen peroxide oxidation current of a cholesterol oxidase sensor on the concentration of cholesterol and sitosterol.

Such transformations can also use 1-hydroxy-1/7-ben-zotriazole in place of the benzothiazoline.159 An alkane hydroxylase has been used to convert alkanes to alcohols.160 Cholesterol can be oxidized to the ketone, cholestenone, with cholesterol oxidase in reversed micelles of water in isooctane in 100% conversion.161... 

However, foUowing enrichment of the cells with exogenous cholesterol or incubation with 0.1 mM chlorpromazine [156], the whole of the cholesterol pool becomes available to the cholesterol oxidase. This means that the time-course of cholesterol oxidation can be determined and hence the rate of flip-flop. Based on such studies, a half-time of less than 3 sec at 37°C has been found for transposition of cholesterol across the bilayers [157]. These studies have also been extended to probe features of lipid-cholesterol organisation in the human erythrocyte membrane [158]. Clearly, such studies are relevant to our understanding of the mechanisms of cholesterol loss from cells in vivo by the methods outhned earlier. 

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