Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.     

Fluorometric determination of histamine with OPT: optimum reaction conditions and tests of identity

Histamine reacts with OPT at an alkaline pH giving fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 40 min reaction at 0°C under continuous gassing with nitrogen. After acidification with sulfuric acid to pH 2–5 the fluorescence was stable for hours. Reagent blanks were reduced by the lowering of the reaction temperature and by decreasing the amount of OPT added. These modifications permitted the determination of 2 ng histamine/ml and gave far better reproducibility than the original procedure of Shore, Burkhalter, and Cohn. The fluorometric assay for histamine is nonspecific; the major interfering OPT-reactive tissue component is believed to be spermidine. Specificity was secured by adding formaldehyde before acidification, thus abolishing the fluorescence of histamine but not that of spermidine, or by adding CdCl₂ or SrCl₂ together with OPT, thus preventing the formation of the spermidine fluorophore but not that of the histamine fluorophore.     

Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide

A simple assay for O-glycosylated glycoproteins involving the liberation of oligosaccharides by beta-elimination with dilute alkali and the subsequent derivatization of the reducing end with 2-cyanoacetamide is reported. The method can be used to quantitate microgram amounts of mucin within 30 min. The assay is 30 times less sensitive to protein or N-linked glycoproteins and 100 times less sensitive to DNA than to the corresponding weight of canine tracheal mucin. Of the substances tested, only cesium chloride and potassium thiocyanate caused substantial interference, but in neither case was this sufficiently serious to prevent the method being used for monitoring mucin purification schemes utilizing these reagents. The coefficients of variation for replicate analyses of canine tracheal mucin (14.0, 5.0, and 2.0 micrograms) were 3.6, 6.5, and 12.3%, respectively.     

Fluorometric determination of carbodiimides with trans-aconitic acid

A new fluorometric method for the determination of carbodiimides with trans-aconitic acid as a reagent has been developed. With the present method, as little as 50 pmol of carbodiimides can be determined. The relative standard deviations obtained with 25 nmol of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) were 1.35, 1.41, and 2.72% (n = 10), respectively.     

Fluorometric determination of carbohydrate with 2-aminothiophenol

The 2-aminothiophenol-based fluorometric assay of Nakano et al. (1973, J. Pharm. Soc. Jpn. 93, 350-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 50 ng of carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates.     

Fluorometric determination of trioses with o-phenylphenol.

A spectrophotometric method for determining ascorbic acid based on the redox reaction between a copper (II)-2,2′-biquinoline solution and ascorbic acid was developed. The purple color of the copper (I)-2,2′-biquinoline complex formed in a buffered acetone-water solution was measured at 540 nm. Minerals, sugars, and other vitamins do not interfere when present in quantities usually found in pharmaceutical preparations. Ferrous interference was eliminated by treating the sample solution with the cation-exchange resin Dowex 50W-X12. Several single component and multivitamin formulations were satisfactorily analyzed by this method. Its high sensitivity permits measurements of quantities of ascorbic acid to 3.4 μg/ml of sample solution. The procedure is simple, rapid, and suitable for routine control.     

Fluorometric detection of adenine in target DNA by exciplex formation with fluorescent 8-arylethynylated deoxyguanosine

We demonstrated an intriguing method to discriminate adenine by incident appearance of an intense new emission via exciplex formation in hybridization of target DNA with newly designed fluorescent 8-arylethynylated deoxyguanosine derivatives. We described the synthesis of such highly electron donating fluorescent guanosine derivatives and their incorporation into DNA oligomers which may be used for the structural study and the fluorometric analysis of nucleic acids.     

Chemiluminescent derivatization of adenyl compounds with glyoxal derivatives in the presence of heteropoly acids and its application to the simple and sensitive determination of DNA

A chemiluminescence (CL) determination of adenyl compounds is described. CL derivatization of adenyl compounds with methylglyoxal dimethyl acetal was performed in the presence of tungstosilicic acid and propan-2-ol. CL from adenyl compounds was produced by hydrogen peroxide and L -cysteine ethyl ester in DMF and water. The proposed method is highly sensitive and specific to compounds containing adenine. Adenine was determined in the range 1.0 × 10⁻³ −5.0 × 10⁻⁸ M with the detection limit of 3.0 × 10⁻⁸ M (150 fmol per assay). The method was applied to the determination of DNA and detection limits of a few nanograms of DNA achieved. © 1998 John Wiley & Sons, Ltd.     

Fluorometric determination of carbohydrates

Fluorometric methods are described for the determination of the enzymes hexokinase and β-glucosidase, and the carbohydrates glucose, fructose, maltose, cellobiose, lactose, glycogen, and salicin Glucose and fructose are determined fluorometrically using hexokinase and the resazurin-resorufin indicator reaction. Maltose, cellobiose, lactose, glycogen, and salicin are enzymically hydrolyzed to glucose, which is then determined fluorometrically using glucose oxidase, p-hydroxyphenylacetic acid, and peroxidase. All the carbohydrates were assayed in the range 0.01–50 μg/ml and the enzymes hexokinase and β-glucosidase in the range 10^?3 to 10^?1 unit/ml with an accuracy and precision of about 1.5%.     

The reaction of horse-liver alcohol dehydrogenase with glyoxal.

Horse liver alcohol dehydrogenase was reacted with glyoxal at different pH values ranging from 6.0 to 9.0. At pH 9.0 the enzyme undergoes a rapid activation over the first minutes of reaction, followed by a decline of activity, which reaches 10% of that of the native enzyme. Chemical analysis of the inactivated enzyme after sodium borohydride reduction shows that 11 argi-ine and 11 lysine residues per mole are modified. At pH 7.7 the enzyme activity increases during the first hour of the reaction with glyoxal and then decreases slowly. Chemical analysis shows that 4 arginine and 3 lysine residues per mole are modified in the enzyme at the maximum of activation. At pH 7.0 the enzyme undergoes a 4-fold activation. Chemical analysis shows that in this activated enzyme 3 lysine and no arginine residues per mole have been modified. Steady-state kinetic analysis suggests that the activated enzyme is not subjected to substrate inhibition and that its Michaelis constant for ethanol is three times larger than that of the native enzyme. The possible role of arginine and lysine residues in the catalytic function of liver alcohol dehydrogenase is discussed.     

Fluorometric determination of spermidine using o-phthalaldehyde: optimum reaction conditions and tests of identity

Spermidine and histamine react with o-phthalaldehyde at alkaline pH, giving rise to fluorescent conjugation products that are stabilized by acidification to pH 2–4. The reaction has been used in the fluorometric assay of both these compounds. In the present study, the reaction conditions have been analyzed with the purpose of improving the sensitivity as well as the specificity of the spermidine assay. The sensitivity was improved by carrying out the condensation at pH 11.0–11.6 for 2 min at 100°C. Under these conditions, the lowest measurable amount of spermidine was 10 ng/ml, and the fluorescence of histamine (below 1–2 μg/ml) was nonmeasurable. Boiling the samples for 1 hr after the acidification did not affect the spermidine fluorescence but abolished residual histamine fluorescence. The spermidine fluorescence failed to develop in the presence of CdCl₂ or SrCl₂, whereas the histamine fluorescence was unaffected. Crude extracts of rat brain gave fluorescence readings similar to those of butanol extracts, suggesting that extensive purification of tissue sperimidine prior to assay is not always necessary.     

Fluorometric determination of deoxyribonuclease I activity with PicoGreen

A rapid and sensitive assay for the detection of deoxyribonuclease I (DNase I) activity is described. This method is based on the ability of PicoGreen dye to enhance its fluorescence when bound to double-stranded DNA. In the standard assay, reaction mixtures containing the DNase I sample and 0.2 microg of the substrate DNA were prepared in a fluorescence microtiter plate and incubated at 37 degrees C. At the end of the reaction, the diluted PicoGreen reagent was added to each well and fluorescence intensity was measured with a fluorescence plate reader. By this assay, it was possible to determine precisely as little as 5 pg of DNase I within an hour. Moreover, using a small amount of the substrate DNA, the method was shown to be suitable for the sensitive detection of DNase I inhibitor activity.     

Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference.     

Hydrolytic cleavage of N6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases

Homogeneous adenine deaminases (EC 3.5.4.2) from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and a putative ADA (adenosine deaminase; EC 3.5.4.4) from Arabidopsis thaliana were obtained for the first time as purified recombinant proteins by molecular cloning of the corresponding genes and their overexpression in Escherichia coli. The enzymes showed comparable molecular properties with well-known mammalian ADAs, but exhibited much lower k(cat) values. Adenine was the most favoured substrate for the yeast enzymes, whereas the plant enzyme showed only very low activities with either adenine, adenosine, AMP or ATP. Interestingly, the yeast enzymes also hydrolysed N6-substituted adenines from cytokinins, a group of plant hormones, cleaving them to inosine and the corresponding side chain amine. The hydrolytic cleavage of synthetic cytokinin 2,6-di-substituted analogues that are used in cancer therapy, such as olomoucine, roscovitine and bohemine, was subsequently shown for a reference sample of human ADA1. ADA1, however, showed a different reaction mechanism to that of the yeast enzymes, hydrolysing the compounds to an adenine derivative and a side chain alcohol. The reaction products were identified using reference compounds on HPLC coupled to UV and Q-TOF (quadrupole-time-of-flight) detectors.The ADA1 activity may constitute the debenzylation metabolic route already described for bohemine and, as a consequence, it may compromise the physiological or therapeutic effects of exogenously applied cytokinin derivatives.