The kinetics of the interaction between the actin‐binding domain of α‐actinin and F‐actin

Measurement of the binding equilibrium for the interaction of α‐actinin with F‐actin is complicated by secondary reactions involving cross‐linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin‐binding domain (ABD) of chick smooth muscle α‐actinin with F‐actin. Stopped‐flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F‐actin yielding second order rate constants for association of 2 × 10 5 , 1.8 × 10 6 and 4 × 10 6 M −1 · s −1 at 5°C, 15°C and 25°C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6 s −1 , giving equilibrium constants of 0.83 and 2.4 μM, respectively. Optical changes on mixing intact α‐actinin with F‐actin were dominated by secondary bundling events.