Figure 3. Binding of TrCel7A to BC is only partially reversible.

(A and B) Dilution experiment. BC at 1.0 g/L was incubated with 1.0 µM TrCel7A to establish equilibrium. Equilibrium was disturbed by the addition of buffer to bring up tenfold dilution and relaxation to new equilibrium was followed. (A) Binding reversibility was assessed in the basis of both, total bound TrCel7A, TrCel7A_Bound and active site bound TrCel7A, TrCel7A_Bound-OA. Dotted lines show the progression from the initial equilibrium to the disturbed equilibrium to the new equilibrium. (B) Change in the concentration of TrCel7A free from cellulose, TrCel7A_Free and TrCel7A with free active site TrCel7A_FA in time after disturbance of equilibrium by dilution. (C) Free enzyme depletion experiment. BC at 0.1 g/L was incubated with TrCel7A at different concentrations to establish equilibrium. Cellulose with bound enzyme was pelleted by centrifugation, 90% of the supernatant was withdrawn and pellet was resuspended in the same amount of buffer to disturb the equilibrium. The position of the new equilibrium was measured after 30 min from the disturbance. Binding reversibility was assessed in the basis of total bound TrCel7A. Dotted lines show the progression from the initial equilibrium to the disturbed equilibrium to the new equilibrium.