FIG. 2.

Equilibrium titration of Ras(D119N) with Cdc25^Mm285. Increasing amounts of Cdc25^Mm285 were added to 86 nM Ras(D119N)-mXppNHp at 20°C in standard buffer, causing a decrease in relative (rel.) fluorescence related to the liberation of the nucleotide. (A) After each addition of Cdc25^Mm285, the fluorescence signal was measured for typically 5 to 10 min in order to ascertain a stable value. At the end of the titration, a 200-fold excess of Xpp(NH)p was added in order to determine the fluorescence of the unbound mXpp(NH)p. (B) Plotting of the fluorescence data as a function of the Cdc25^Mm285 concentration and fitting of these data with the multiparameter program Fascimile as described by Lenzen et al. (23). x, experimental data; +, calculated data.