Quantitative measurements nucleotides

Adsorption onto activated charcoal (Norit A) has been used for small oligonucleotides, up to tetranucleotides (Crane and Lipman 1953 Mandeles and Kammen 1966). The method is quicker and more convenient than DEAE-cellulose chromatography. Mandeles and Kammen (1966) used Norit A suspended in 1 mM phosphate 1 mM pyrophosphate buffer at pH 6.0 at a concentration of 100 mg/ml. The nucleotide solution is adjusted to pH 3 and 5 mg Norit for every optical density unit ( 40 pg) of nucleotides is added. The charcoal is collected on a filter paper and washed with water. (A good quantitative measure of or radio-activity may be obtained by counting the dry filter paper in a Geiger-Muller thin window counter.) The nucleotide material is eluted with a small volume of a mixture of ethanol, water and ammonia (600 400 6.5 v/v/v) which is then removed by drying under reduced pressure at 40°C. 

Figure 2 DNA damage induced by ionizing radiation. A) DNA damage and repair. All the constitutive elements of DNA (sugar-phosphate backbone and bases) are possibly modified by ionizing radiation. Single strand breaks (SSB), oxidized bases and abasic site are processed by base excision repair (BER), double strand breaks (DSB) by homologous recombination and non homologous end joining (HR and NHEJ) and DNA-protein crosslinks by nucleotide excision repair (NER). B) Quantitative measurement of radiation-induced and spontaneous DNA damage.

In order to make quantitative measurements. Miles (1958f)) has determined integrated infrared absorption intensities for some nucleosides, nucleotides, and polynucleotides in DjO solution (Fig. 12.8 and Table 12.2) by application of Ramsay s method I (Ramsay, 1952). When there is a well-resolved band, the application of Ramsay s method encounters no difficulty, but when overlapping bands occur, as in uridine and its derivatives, some uncertainty exists in determining the halfband width of a particular band. (The half-band width is a factor in Ramsay s equation and is defined as, Av, 2 = the width of the band in cm" at half-maximum intensity.) The equation as used by Miles to obtain A, the true integrated absorption intensity... 

For individual nucleotides isolated by chromatographic procedures, the ultraviolet absorption spectra provide facile identification and quantitation (/). Well-known chemical (colorimetric) methods are available to identify and quantitate the sugar component, and to measure the relative amounts of sugar and phosphate in isolated nucleotide fractions (34). Enzymatic methods are also used in characterizing and in quantitation of nucleotides (35). 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Little quantitative work has appeared on the determination of the rate of conversion of the various purine analogues to their nucleotides with highly purified phosphoribosyltransferases from mammalian cells or any other source, but there would appear to be a rough correlation between the cytotoxicity of these analogues and their ability to serve as substrates (the Kj values for a number of purines and purine analogues have been determined [45a, 85a] but this value is not a measure of conversion to nucleotide). Table 2.1 lists a number of purine analogues and an estimate of their ability to serve as substrates for the phosphoribosyltransferases. 

A high-throughput assay for bacterial RNA polymerase has been successfully developed and validated using a 96-well, automated format [70], The reaction mixture contained a DNA template, nucleotide substrates (NTPs), supplemented with a-33P-labeled CTP in Tris-acetate buffer (pH 6.8). The polymerase reaction was carried out at 34°C for 40 min (providing linear kinetics). The effect of dimethylsulfoxide (DMSO), the usual solvent for test compounds used in a screen, was taken into consideration. The radiolabeled RNA transcripts were allowed to bind diethyl aminoethyl (DEAE) beads, which were then separated via filtration, and radioactivity associated with the wells was quantitated to measure the RNA polymerase activity. The standard deviation of the measured activity was typically < 15% of the average. Use of this assay to screen for RNA polymerase inhibitors from chemical libraries and natural products led to the identification of DNA intercalators (known to inhibit RNA polymerase activity), rifampicin (a known inhibitors of RNA polymerase), and several derivatives of rifampicin from Actinomycetes extracts. Therefore this assay can be reliably utilized to detect novel inhibitors of bacterial RNA polymerase. 

Pruvost, A. et al. Specificity enhancement with LC-positive ESl-MS/MS for the measurement of nucleotides apphcation to the quantitative determination of carbovir triphosphate, lamivudine triphosphate and tenofovir diphosphate in human peripheral blood mononuclear cells. J. Mass Spectrom. 2008,43, 224—233. 

Figure 7 shows the response of the redox potential in a perfused hamster liver to the addition of 45 mN ethanol. Instead of the vitro labeling strategy Just described. the pyridine nucleotide pools in this hamster liver were labeled vivo by intraperitoneal injection of 35 mg [5- C] nicotinamide 5 hours prior to sacrifice. The bottom two spectra (2.6 min and 12.8 min) were obtained prior to addition of ethanol. They show resonances from labeled NAD, natural abundance glycogen and natural abundance choline methyl groups of phospholipids but no resonance from reduced pyridine nucleotides. After addition of 45 mM 10% [1- C] ethanol (at 17.9 min), resonances from C-1 of ethanol and NADH are detectable. These data demonstrate that the pyridine nucleotide pools labeled by intraperitoneal injection are metabolically active and that addition of 45 mN ethanol results in a marked change in the redox potential of the liver as measured by NNR. Furthermore, the observation of separate resonances for the oxidized and reduced pyridine nucleotides indicates that chemical exchange between oxidized and reduced forms is slow on the NNR time scale, and demonstrate that NNR may be used to quantitate the redox potential of free pyridine nucleotides situ. 

A major factor complicating the quantitative interpretation of most adenylate cyclase measurements is contamination by other enzymes including ATPase, inorganic pyrophosphatase, cyclic nucleotide phosphodiesterase, and various deaminases. Some degree of inhibition of phosphodiesterase is necessary in most preparations, and a methylxan-... 

The spots can be seen in near ultraviolet light in a dark room because they quench the fluorescence of the paper. (Wear protective goggles when using ultraviolet light). For quantitative analysis the spots are cut out, the nucleotides eluted with water or dilute buffer or 0.05 N HCl and their optical densities measured. The extinction coefficients at pH 7 are given in Table 2.1. 

Single-stranded DNA containing deoxyguanosine residues can be quantitated in the presence of terbium(III). Ten micromolar Tb3+ in a pH 6 cacodylate buffer shows no detectable fluorescence, with excitation at 290 nm and emission measured at 488 nm. In the presence of single-stranded DNA, Tb3+ coordinates with deoxy-guanosine-5-phosphate nucleotides, and a linear dependence of fluorescence on concentration has been observed over the 1-10-pg/mL range of thermally denatured rat liver DNA.19... 

Whereas detection at one wavelength may suggest a pure zone, an Interference will be detected only at the other wavelength. If no choice can be made as to which wavelength Is best for a certain class of compounds, such as nucleotides, a ratio plot Includes the Information of both wavelengths. Quantitation by measuring zone lengths can also be applied to the ratio plot. Purther data reduction by the micro computer is possible. 

Purine analysis can be achieved with this system, specifically inosine (the co-substrate, with phosphate. Scheme 5.5) can be determined quantitatively using similar methodology. The hypoxanthine ratio ([hypoxanthine]/ [hypoxanthine] + [inosine] + [inosine-monophosphate]) is key measure of fish freshness as hypoxanthine is a product of nucleotide degradation and a sign of spoilage. As mentioned previously, hypoxanthine or xanthine... 

The Gp Rh-based sensor can also be used for quantitative analyses. This was demonstrated with the analytes ATP, cAMP, and PPi. The hydrolysis of ATP to give cAMP and PPi is catalyzed by adenylate cyclase (AC). AC is an important enzyme that is involved in many signaling pathways. Its activity is commonly measured by monitoring the conversion of [a- P]ATP to [a- P]cAMP using ion-exchange chromatography to separate the nucleotides. To show that the sensor can be used to measure simultaneously the concentrations of ATP and cAMP/PPi,... 

---