Fig. 3.

Enzyme-coupled assay for formation of dG from dGTP. The reaction contained dGTP substrate, Dgt enzyme, Purine Nucleoside Phosphorylase and Xanthine Oxidase, as described in Materials and Methods. The reaction was monitored by increase in absorbance at 297 nm. The concentration of dGTP was varied from 2 to 120 µM. Panels A and B show the reactions for the 2–20 µM and 25–120 µM range, respectively. For each reaction, the zero-order reaction velocity (continuous black lines) was determined and used to derive the kinetics parameters of Fig. 4B. The lag-time before steady-state intermediate concentrations are reached, as observed here, is well known for enzyme-coupled assays [29].