Figure 5. Titration of the cytomegalovirus DNA polymerase with dATP.

(A) Increasing concentrations of dATP were added to a 1 μM solution of the enzyme in binding buffer (20 mM Tris/HCl, pH 7.5, 50 mM NaCl and 10 mM MgCl₂) and the emission spectrum was scanned from 310 to 440 nm. A double-reciprocal plot of the saturation isotherm is shown in the right-hand corner. (B) The effect of increasing ionic strength on the apparent dissociation constant of the enzyme for dATP was investigated. Increasing concentrations of KCl were added to the binding reactions to generate the desired ionic strengths. (C) Thermodynamic parameters of the interaction between dATP and the cytomegalovirus DNA polymerase. Binding reactions were performed at various temperatures, and the respective association constants were evaluated. A van't Hoff plot for the interaction between dATP and the protein is shown. The effect of temperature on the association constant was evaluated at pH 7.5. (D) Kinetic analysis of real-time binding of dATP to the protein. A 1 μM solution of the enzyme was incubated with 1 mM dATP. Emission was monitored for 30 s at 338 nm, and excitation was performed at 290 nm.