Charge effects on the dynamic quenching of fluorescence of 1,N6-ethenoadenosine oligophosphates by iodide, thallium (I) and acrylamide.

Systematic studies were carried out to determine how the dynamic quenching of fluorescence was affected by electrostatic interaction between charges carried by the fluorophore and the quencher. 1,N6-Ethenoadenosine oligophosphates (epsilon-ATP, epsilon-ADP, epsilon-AMP, and epsilon-Ad) were used as fluorophores; these compounds have the same luminous group (epsilon-adenine ring) with variously charged phosphate groups. Acrylamide and the iodide (I-), well known to be effective quenchers carrying zero and one negative charge, respectively, were used as quenchers. The thallium (I) (Tl+) was also found to be an effective quencher as a result of studies of the quenching activity of various positively charged ions. Results which agreed qualitatively with those expected were obtained for the charge effects on the dynamic quenching and the effects of ionic strength on it. That is, the repulsive force exerted between I- and phosphate groups decreased the quenching rate, and increasing ionic strength of solutions mitigated these charge effects. The results obtained, however, were not quantitatively consistent with theoretical expectations for ionic reaction rates in solutions. One reason for the quantitative disagreement is that the negatively charged phosphate groups are located at a distance from the luminous group. By theoretical analysis of the quenching data, the distance was calculated to be 9.6 A + delta r, where delta r changes according to the number of phosphate groups. Furthermore, through a theoretical analysis of the disagreement, we found a new factor, the "size effect" of a luminous group, which modifies the charge effects on dynamic quenching. The size effect provides information on the gradient of the electric potential generated by the phosphate groups. These studies provide a basis for the study of electric potential at local regions of proteins.