The Tb3+ binding to spectrin tetramer (SPT) was studied by Tb3+ fluorescence titration and CD spectra. The results indicated that the total high-affinity Tb3+ binding sites are n1 = 330, with average Kd = 3.6 x 10(-6) M. Among them, ca. 90 sites are of higher affinity and are probably more specific to Tb3+ than the remaining sites. There are 520 low affinity Tb3+ binding sites with average Kd = 1.5 x 10(-5) M. Fluorescence and CD spectra revealed that the alpha-helix content of SPT decreased with Tb3+ binding to specific sites and further binding did not result in conformation change. Tb3+ binding to SPT and the subsequent reactions were studied by employing stopped-flow fluorescence and light scattering methods. The studies demonstrate that this is a multistep reaction assembly: high-affinity terbium binding-conformation change-aggregation-low-affinity terbium binding--the second conformation change. The critical Tb3+ concentration-induced spectrin dimer (SPD) aggregation was determined with a light scattering method.