Stability of the complex between HMG1 and Form X as a function of time.(A) Complexes between labelled Form X and HMG1 were first formed during a 30 min incubation at 37° (lane 0, concentration of Form X = 10-11 M; concentration ratio [HMG1]/[FormX] ≅ 2). At time 0, a 40 fold excess of unlabelled Form X was added and incubation at 37° was resumed. At each indicated time, from 1 min to 120 min, a sample was taken from the incubation mixture and immediately loaded on a running polyacrylamide gel, thus freezing the dissociation process. The curved shape of the autoradiogram is due to the fact that the first samples have migrated 4 hr while the last loaded samples have migrated 2 hr only. Controls: C, Form X with no protein added; ∞, competitor was added before the protein, which mimics complete protein redistribution after an incubation for an infinite time. (B) The radioactivity in the bands in (A) was counted with a phosphorimager and plotted as a function of time. Squares and green line: complex C2; triangles and blue line: complex C1; circles and red line: free Form X. (C) Best fit obtained by simulating the experiment with the program Chemical Kinetics Simulator, yielding estimates of for the dissociation time constants koff equal to 1.7 × 10-4 s-1 for complex C1 and 1.2 × 10-3 s-1 for complex C2.