The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.
Several solution properties of complexes formed between the trivalent lanthanide ions (LnIII) and the macrocyclic ligand DOTP8-, including stability constants, protonation equilibria, and interactions of the LnDOTP5- complexes with alkali metal ions, have been examined by spectrophotometry, potentiometry, osmometry, and 1H, 31P, and 23Na NMR spectroscopy. Spectrophotometric competition experiments between DOTP and arsenazo III for complexation with the LnIII ions at pH 4 indicate that the thermodynamic stability constants (log K ML) of LnDOTP5- range from 27.6 to 29.6 from LaIII to LuIII. The value for LaDOTP5- obtained by colorimetry (27.6) was supported by a competition experiment between DOTP and EDTA monitored by 1H NMR (27.1) and by a potentiometric competition titration between DTPA and DOTP (27.4). Potentiometric titrations of several LnDOTP5- complexes indicated that four protonation steps occur between pH 10 and 2; the protonation constants determined by potentiometry were consistent with 31P shift titrations of the LnDOTP5- complexes. Dissection of the 31P shifts of the heavy LnDOTP5- complexes (Tb → Tm) into contact and pseudocontact contributions showed that the latter dominated at all pH values. The smaller 31P shifts observed at lower pH for TmDOTP5- were partially due to relaxation of the chelate structure which occurred upon protonation. The 31P shifts of other LnDOTP5- complexes (Ln = Pr, Nd, Eu) showed a different pH-dependent behavior, with a change in chemical shift direction occurring after two protonation steps. This behavior was traced to a pH-dependent alteration of the contact shift at the phosphorus nuclei as these complexes were protonated. 23Na NMR studies of the interactions of TmDOTP5- with alkali and ammonium cations showed that Et4N+ and Me4N+ did not compete effectively with Na+ for the binding sites on TmDOTP5-, while K+ and NH4 + competed more effectively and Cs+ and Li+ less effectively. A 23Na shift of more than 400 ppm was observed at low Na+/TmDOTP5- ratios and high pH, indicating that Na+ was bound near the 4-fold symmetry axis of TmDOTP5- under these conditions. Osmolality measurements of chelate samples containing various amounts of Na+ indicated that at high Na+/TmDOTP5- ratios at least three Na+ ions were bound to TmDOTP5-. These ions showed a significantly smaller 23Na-bound shift, indicating they must bind to the chelate at sites further away from the 4-fold symmetry axis. Fully bound 23Na shifts and relaxation rate enhancements and binding constants for all Na x H y TmDOTP species were obtained by fitting the observed 23Na shift and relaxation data and the osmometric data, using a spreadsheet approach. This model successfully explained the 23Na shift and osmolality observed for the commercial reagent Na4HTmDOTP·3NaOAc (at 80 mM at pH 7.4).
A simple and sensitive NMR method for quantifying excess 13 C-enrichment in positions 2 and 3 of lactate by 1 H NMR spectroscopy of the lactate methyl signal is described. The measurement requires neither signal calibrations nor the addition of a standard and accounts for natural abundance 13 Ccontributions. As a demonstration, the measurement was applied to ϳ3 mol of lactate generated by erythrocyte preparations incubated with [2-13 C]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PP fluxes were estimated from the ratio of excess 13 C-enrichment in lactate carbon 3 relative to carbon 2 in accordance with established metabolic models. Under baseline conditions, PP flux accounted for 7 ؎ 2% of glucose consumption while in the presence of methylene blue, a classical activator of PP activity, its contribution increased to 27 ؎ 10% of total glucose consumption (P < 0.01 In erythrocytes, the sacrificial oxidation of reduced glutathione (GSH) is the primary defense against oxidative stress. Under these conditions, GSH is regenerated by the NADPH-dependent glutathione reductase, while NADPH is replenished via the oxidation of glucose through the oxidative branch of the pentose phosphate pathway (PP). In response to an oxidative insult, PP flux is increased to the extent that it accounts for a significant fraction of glucose consumption. Since NADPH generation via PP is dedicated to the glutathione redox cycle, measurement of PP flux provides a sensitive and direct reflection of erythrocyte glutathione antioxidant activity.For systematic applications, such as screening an array of compounds for oxidant activity, PP flux measurement has to be fast, robust, and reproducible. While many different tracer methods have been developed to quantify the fraction of glucose consumed by PP relative to glycolysis (1-5), it is generally agreed that tracing the label from [2-13 C] or [2-14 C]glucose into positions 2 and 3 of lactate is in theory the optimal approach. The main impediment to this method has been the quantification of positional 14 Cspecific activity or 13 C-enrichment of lactate. With [2-14 C]glucose, determination of the specific activity in carbons 2 and 3 of lactate requires carbon-by-carbon degradation and is therefore not practical for large-scale applications. Lactate 13 C-enrichment from [2-13 C]glucose can be quantified by gas-chromatography/mass spectrometry (GC-MS) but the analysis does not provide positional 13 Cenrichment information (3). Alternatively, NMR analysis of lactate 13 C-enrichment from [2-13 C]glucose can be performed either in situ or following minimal sample manipulation while directly providing positional 13 C-enrichment information. Previous NMR methods have focused on quantifying 13 C-enrichment of lactate by 13 C NMR spectroscopy (6 -9). This approach has low sensitivity, requires corrections for different T 1 and nOe parameters of the lactate carbons, and the natural abundance 13 C contribution (or excess 13 C-enrichment) cannot be directly ...
Ab initio and density functional theory calculations were performed on small Pd clusters to assess their precise energy level characteristics. The ground states of Pd and Pd 3 are found to be singlets while Pd 2 and Pd 4 are triplets. Pd 2 is found to be a weak dimer with bond energy of 18 kcal/mol. The trimer is triangular and the tetramer is of tetrahedral geometry. A nonadditive effect can be observed as the size of cluster increases. Larger clusters are bonded better than smaller ones. The second lowest state of Pd 4 is a singlet of tetrahedral geometry. Modern DFT methods yield results of better quality than sophisticated standard ab initio methods, thereby providing an affordable avenue for the analysis of larger clusters and potential nanoelectronics probes.
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