The physical sizes of F-actin and actin-binding proteins place restrictions on
the types of assays that can be used for measuring equilibrium binding. This
chapter describes variations of sedimentation and fluorescence methods that
have broad applications to the study of actin. Sedimentation methods commonly
consist of two steps: separation of free ligand from the ligand–actin
complex by sedimentation and determination of the free and bound ligand.
Fluorescence methods are based on the change in environment of tryptophan
residues or covalently attached probes on either actin or the ligand protein.
Pyrene on Cys 374 of actin is responsive to binding of myosin subfragment 1
(S1) and other proteins and changes the affinity of binding to S1 by less
than a factor of 2 (1). Fluorescein-labeled S1, 4-(iodoacetamido) salicylic
acid (ISAL)-labeled S1, and N-((2-(iodoacetoxy)ethyl)-N-methyl)-amino-7-
nitrobenz-2-oxa-1,3-diazole (IANBD)-labeled caldesmon are examples of fl uorescent
ligand proteins that have been used in actin-binding studies (2).