Binding of a denatured heme protein and ATP to erythroid spectrin

Spectrin is a large, warm-like cytoskeletal protein that is abundant in all cell types. The denatured heme enzyme, horseradish peroxidase showed signi ficant decrease in the reactivation yield, after 30 min of refolding, in pr esence of increasing concentrations of spectrin from that in the absence, T his indicated that spectrin could bind denatured HRP and inhibit their refo lding. In presence of 1 nM ATP and 10 mg MgCl2 the spectrin binding of dena tured HRP is abolished. This activity of decreasing the reactivation yield was found to be ATP-dependent and the denatured enzyme after 30 min refoldi ng in the presence of spectrin, pretreated with Mg/ATP, showed about 40% in crease in the reactivation yield compared to the same in absence of spectri n. Fluorescence spectroscopic studies indicated binding of ATP to native sp ectrin showing concentration-dependent quenching of tryptophan fluorescence by ATP. The apparent dissociation constant of binding of ATP to spectrin w as estimated to be 1.1 mM. A high affinity binding of spectrin with denatur ed HRP has been characterized (K-d = 16 nM). Since these properties are sim ilar to those of established molecular chaperone proteins, these data indic ate that spectrin might have a chaperone-like function in erythrocytes, (C) 2001 Academic Press.