TB3+ BINDING TO HUMAN ERYTHROCYTE SPECTRIN RESULTING IN CONFORMATION CHANGE AND AGGREGATION

The Tb3+ binding to spectrin tetramer (SPT) was studied by Tb3+ fluore scence titration and CD spectra. The results indicated that the total high-affinity Tb3+ binding sites are n(1) = 330, with average K-d = 3. 6 x 10(-6) M. Among them ca. 90 sites are of higher affinity and are p robably more specific to Tb3+ than the remaining sites. There are 520 low affinity Tb3+ binding sites with average K-d = 1.5 x 10(-5) M. Flu orescence 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 subsequ ent reactions were studied by employing stopped-flow fluorescence and light scattering methods. The studies demonstrate that this is a multi step reaction assembly: high affinity terbium binding-conformation cha nge-aggregation-low-affininity terbium binding-the second conformation change. The critical Tb3+ concentration-induced spectrin dimer (SPD) aggregation was determined with a light scattering method.