Spectroscopic Properties of 2‘-(or-3‘)-O-(2,4,6-Trinitrophenyl) Adenosine 5‘-Triphosphate Revealed by Time-Resolved Fluorescence Spectroscopy

Abstract

We examined the spectroscopic properties of a fluorescent nucleotide analogue, 2‘-(or-3‘)-O-(2,4,6-trinitrophenyl) adenosine 5‘-triphosphate (TNP-ATP), using time-resolved fluorescence spectroscopy. Our study clearly shows that the change in fluorescence intensity of TNP-ATP with different solvent properties has disparate origins, which were obscured in previous steady-state measurements. The four main findings are as follows. First, the fluorescence lifetime of TNP-ATP was found to increase with increasing viscosity, resulting in an increase in quantum yield, and therefore, fluorescence intensity. Second, fluorescence intensity increased with increasing pH ranging from 2.4 to 8.1. However, the fluorescence lifetime was essentially independent of the change in pH. The change in fluorescence intensity was not due to variations in quantum yield, but to the change in absorbance with pH. Third, we examined the interaction of TNP-ATP with the Klenow fragment of DNA polymerase I and found that free TNP-ATP and bound TNP-ATP in two binding sites were distinguished by using time-resolved spectroscopy, although the binding site with lower affinity could not be resolved in previous steady-state measurements. As the enzyme concentration was increased, the fluorescence lifetime of the enzyme−TNP-ATP complex remained unchanged, but the fractional fluorescence intensity of the bound TNP-ATP increased, thus leading to an increase in total fluorescence photocounts. Last, the fluorescence of TNP-ATP in the presence of the enzyme was further enhanced by the addition of Mg2+. This enhancement was due to the increase in the fractional fluorescence intensity of the bound TNP-ATP rather than to the change in fluorescence lifetime. This finding indicates that Mg2+ raises the affinity of the enzyme for TNP-ATP.