Figure 2.

Alternative methods to determine association and dissociation rate constants. (A) Association experiments with HeLa cells were performed as in Fig. 1 by using various ¹²⁵I-TNF concentrations (100, 200, 400, 800, and 1,600 pM) (Inset). The obtained net association rate values were plotted against the ¹²⁵I-TNF concentration initially present in the assay to determine the dissociation rate constant (ordinate intercept) and the association rate constant (slope). Shown is a representative experiment (n = 3). (B) HeLa cells were incubated at 37°C with 250 pM ¹²⁵I-TNF to obtain the association kinetics shown (▪). After 2, 5, and 7 min of incubation (arrows), aliquots were supplemented with a 200-fold excess of unlabeled TNF in prewarmed binding buffer, and cell-bound radioactivity was followed for an additional 7 min (○). The dotted curves passing through the data represent the calculated dissociation kinetics based on the dissociation rate constants obtained from the experiments shown in Fig. 1.