Abstract
The low fluorescence yield of nucleic acids makes it necessary either to attach extrinsic fluorophores, or add fluorescent intercalators in the case of dsDNA. We have found that the precence of 3-bromopropan-1-ol enhances the fluorescence yield of adenine, adenosine, 6-methylpurine and 7-methyladenine. In contrast, guanine, hypoxanthine, cytosine and poly-Adenosine did not exhibit this effect. This is due to an apparent shift in pKa of these molecules. In this work, we will focus our attention on adenine. Monitoring fluorescence from adenine as a function of 3-bromopropan-1-ol concentration, we constructed a Benesi-Hildebrandt plot that revealed the formation of a 1:1 complex with an equilibrium constant and Gibbs free energy of K = 1.7E-5 and ΔGo = −28.7 kJ/mol, respectively. We determined the fluorescence yield of adenine to increase about two orders of magnitude once the complex is formed. A second aspect of our work was to explore practical applications of this phenomenon. The observation that hypoxanthine was not similarly fluorescence enhanced allowed us to observe the kinetics deamination of adenine catalyzed by the enzyme adenosine deaminase (ADA). The reaction involves the exchange of an amino group for a hydroxyl group. The standard assay for ADA relies on the difference of absorption measurements. This standard assay is of limited sensitivity, since the absorption spectra of the substrate and product are overlapping, and the magnitude of their extinction coefficients are similar. The method we are developing relies on fluorescence spectroscopy, which proves to be more sensitive and exclusively detects adenine. Via this method we were able to study the kinetics of this reaction and determine the Michaelis constant and Vmax. The production of hypoxanthine was confirmed using HPLC separation techniques.
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