Flavin adenine dinucleotide analysis

HPLC with fluorescence detection was employed for the analysis of riboflavin (RF), flavin mononucleotide (FMN) and flavin-adenin dinucleotide (FAD) in beer, wine and other beverages. The investigation was motivated by the finding that these compounds are responsible for the so-called taste of light which develops in beverages exposed to light. Samples were filtered and injected in to the analytical column without any other pretreatment. Separations were carried out in an ODS column (200 X 2.1mm i.d. particle size 5 pm). Solvents A and B were 0.05 M phosphate buffer (pH 3) and ACN, respectively. The... 

Abbreviations FAD, flavin adenine dinucleotide Fe-S, iron-sulfur proteins that can he identified in separate clusters by electron paramagnetic resonance analysis (the s-1, s-2 subscripts identify these iron-sulfur proteins as part of the succinate dehydrogenase complex) His, the histidine linkage between FAD and the large (70,000 daltons) protein moiety of the enzyme FMN, flavin mononucleotide N-la, N-2 subscripts identify these iron-sulfur proteins as part of the NADH-dehydro-genase complex UQ, ubiquinone Cyt bf and Cyt b, cytochrome b-566 and b-563, respectively. 

ADP AFP ab as ALAT AP ASAT ATP BQ BSA CEH CK CME COD con A CV d D E E EC ECME EDTA EIA /e FAD FET FIA G GOD G6P-DH HBg HCG adenosine diphosphate a-fetoprotein antibody antigen alanine aminotranferase alkaline phosphatase aspartate aminotransferase adenosine triphosphate benzoquinone bovine serum albumin cholesterol ester hydrolase creatine kinase chemically modified electrode cholesterol oxidase concanavalin A coefficient of variation (relative standard deviation) layer thickness diffusion coefficient enzyme potential Enzyme Classification enzyme-chemically modified electrode ethylene diamine tetraacetic acid enzyme immunoassay enzyme loading factor flavin adenine dinucleotide field effect transistor flow injection analysis amplification factor glucose oxidase glucose-6-phosphate dehydrogenase hepatitis B surface antigen human chorionic gonadotropin... 

Several other analytical procedures are available for enzyme activity determination. Fluorescence, this is the ability of certain molecules to absorb light at a certain wavelength and emit it at another, is a property than can be used for enzymatic analysis. NADH, but also FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) have this property that can be used for those enzyme requiring that molecules as coenzymes (Eschenbrenner et al. 1995). This method shares some of the good properties of spectrophotometry and can also be integrated into an HPLC system, but it is less flexible and the equipment not so common in a standard research laboratory. 

This is one of the few oxidoreductases which is conventionally used in the food industry and also in chemical analysis (see section 1.5). The enzyme catalyzes the oxidation of glucose to gluconolactone (that can spontaneously yield gluconic acid) by molecular oxygen which in the presence of water is reduced to hydrogen peroxide. The enzyme (E) requires the coenzyme flavin adenine dinucleotide (FAD) which acts as the electron transporter, according to ... 

Vitamin B2 Food contains three B2 vitamers, riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, which are the predominant vitamers in foods and are usually bound to proteins. Their analysis usually takes place after extraction with dilute mineral acids with or without enzymatic hydrolysis of the coenzymes (which is necessary to convert all forms to riboflavin and to quantify them as total riboflavin). The extracts may be purified using SPE with Cig cartridges. All the operations performed prior to analysis need to be done under subdued lighting to avoid decomposition of riboflavin upon exposure to light. RP chromatography with Cig columns is used along with fluorescence detection (excitation, 440 nm emission, 520 nm). 

Hampel, D., York, E., and Allen, L. 2012. Ultra-performance hquid chromatography tandem mass-spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk. J. Chromatogr. B. 903 7-13. 

Riboflavin (vitamin B2) also acts as a cofactor and is a precursor for the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes are used in metabolism and catalyze numerous oxidation—reduction reactions. Among the good dietary sources for riboflavin, most animal-derived products, milk and dairy products, are pointed out. Foods are usually pretreated before analysis of riboflavin following similar procedures to those described for vitamin Bi. Similarly, fluorescence detection is mostly employed (370 nm ex., 520 ran em.) after RP separation. 

The degree of enzyme purity will ultimately affect fuel cell performance, particularly when enzyme preparations are used to form immobilized films on electrode surfaces in DET reactions. Contaminating proteins that do not provide electron transfer effectively foul the electrode. When enzyme immobilization techniques are specific to the enzyme, then enzyme purity may not be as much as an issue, but rarely the immobilization technique is absolutely specific to the cathodic or anodic enzyme. For example, an attractive immobilization strategy is to link a particular enzyme to an electrode via its cofactor (e.g., flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), etc.) [59]. The cofactor is linked to the electrode material first and then the apoenzyme is allowed to naturally bind to the cofactor all other proteins in the enzyme preparation that cannot bind the cofactor remain unbound and can be removed. Enzymes used in fuel cells are not so unique, and proteins in the immobilizing preparation may use the same cofactor but not the same fuel during fuel cell analysis or operation. 

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