Dinucleotide analysis

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

Indicators There are certain compounds that are suitable as indicators for sensitive and specific clinical analysis. Nicotinamide adenine dinucleotide (NAD) occurs in oxidized (NAD" ) and reduced (NADH) forms. Nicotinamide adenine dinucleotide phosphate (NADP) also has two states, NADP" and NADPH. NADH has a very high uv—vis absorption at 339 nm, extinction coefficient = 6300 (M cm) , but NAD" does not. Similarly, NADPH absorbs light very strongly whereas NADP" does not. 

Naphthalene-2,3-dicarboxaldehyde Nicotinamide adenine dinucleotide N-Acetylneuraminic acid 4-Fluoro-7-nitrobenzoxadiazole Naphthalene-2,3-dicarboxaldehyde Nondestructive readout Near infrared Near infrared fluorescence Nuclear magnetic resonance 2-Nitrophenyl oxalate 1,1 -Oxalyldiimidazole Polycyclic aromatic hydrocarbon Principal component analysis Photosensitized chemiluminescence Pentachlorophenyl oxalate Polymerase chain reaction... 

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... 

The first biochemical analysis of a selenium-containing XDH was reported in 1999 by Andreesen s group. This preparation was specific for xanthine and did not hydroxylate nicotinic acid. Moreover, the enzyme contained FAD, acid-labile sulfur, iron, and a dinucleotide molybdenum cofactor. Most intriguing was the near-equimolar presence of tungsten and molybdenum. It should be noted that the culture medium contained nearly equimolar levels of these metals, making one wonder whether the specificity of this enzyme for metal may be relaxed (i.e., can use Mo or W). Selenium was also found in the preparation and could be released by treatment with cyanide indicating it was also a labile cofactor. This further confirmed the chemical nature of the cofactor from the NAH enzyme from the same strain. ... 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

An enzyme reactor with immobilized 3 -hydroxysteroid dehydrogenase has been successfully used for the analysis of residues of 17 -methyltestosterone in trout by high-performance liquid chromatography (HPLC) (269). Following their separation by reversed-phase chromatography, the major tissue metabolites of 17 -methyltestosterone, namely 5 -androstane-17 -methyl-3, 17 -diol, and 5 -androstane-17 -methyl-3, 17 -diol, were enzymatically modified in the presence of a coreactant, nicotinamide-adenine dinucleotide (NAD), to the corresponding ketone. The position at 3 was enzymatically oxidized, and NADH, the reduced form of NAD, was produced as a coproduct and subjected to fluorescence detection. Reoxidation of NADH to NAD provides the possibility for electrochemical detection. 

As said above, only a summary of the many reported studies can be given here, and for details the reader is referred to the reviews (49-53) and the papers of the groups mentioned in Section III, B (55). A complete conformational NMR analysis of the solution structure of the adduct with a dinucleotide, i.e. cis-Pt(NH3)2 d(GpG)-N7(l),N7(2) ], has been available for some time (50b). The main features of this structure can be summarized as follows ... 

All potentials vs. screen-printed Ag/AgCl pseudo-reference, except values marked with asterisk ( ), which are vs. Ag/3M AgCl double-junction reference electrode, and values marked with dagger CfO, which are vs. saturated calomel. Abbreviations CoPC cobalt phthalocyanine, SPCE screen-printed carbon electrode, GOD glucose oxidase, MWCNT multi-walled carbon nanotubes, NAD nicotinamide adenine dinucleotide, PQQ pyrroloquinoline quinone, FIA flow injection analysis. 

A very interesting observation, first made by Chottard et al.70), deals with the occurrence of conformational isomers after chelation of cisplatin to a number of dinucleotides. In certain cases these isomers are rapidly interconverted, but in many cases they are easily separated by chromatographic techniques, and allow characterisation by NMR techniques70,72 75,92 For such an analysis, application of high-field NMR in conforma-... 

When the dehydrogenases are used in analysis the method relies on measuring the change in the redox state of the cofactor, i.e. the change in the concentration of NAD or NADH. NADH is inherently more easily detected photometrically and electrochemically (see below) than its oxidized counterpart, NAD+ 13). When catalyzed by a dehydrogenase, the redox reaction of the nicotinamide adenine dinucleotides (NAD(P)+/NAD(P)H) is reversible, see Figure 1. A reaction catalyzed by a dehydrogenase can be schematically written as follows ... 

The 3 end of an intron and the 5 end of an exon carry a consensus sequence of CAG G, where the vertical line represents the intron/exon boundary. The AG dinucleotide is scanned from the branch point and the first AG is recognized as the 3 end of the intron (Chen et al 2000). In a patient with congenital myasthenic syndrome, we identified duplication of a 16-nt segment comprised of 8 intronic and 8 exonic nucleotides at the intron 10/exon 10 boundary of CHRNE encoding the acetylcholine receptor epsilon subunit (Ohno et al 2005). We found that the upstream AG of the duplicated segment is exclusively used for splicing and that one or two mutations in the upstream BPS had no effect whereas complete deletion of the upstream BPS partially activated the downstream AG. Similar exclusive activation of the upstream AG is reported in HEXB (Dlott et al., 1990) and SLC4A1 (Bianchi et al, 1997). Creation of a cryptic AG dinucleotide close to the 3 end of an intron should be carefully scrutinized in mutation analysis. 

---