Binding of hemin, hematoporphyrin, and protoporphyrin with erythroid spectrin: fluorescence and molecular docking studies.

Abstract

Free heme has toxic effects, for example lipid peroxidation, DNA damage, and protein aggregation. In severe hemolysis, which occurs during pathological states, for example sickle cell disease, ischemia reperfusion, and malaria, levels of free heme increase inside erythrocytes. The purpose of this study was to investigate whether spectrin, the major erythroid cytoskeleton protein, is involved as an acceptor of free heme. We compared the interactions of three heme derivatives, hemin chloride, hematoporphyrin, and protoporphyrin-IX, with dimeric and tetrameric spectrin. The dissociation constants (K d) for binding to spectrin dimer and tetramer were 0.57 and 1.16 µM respectively. Thermodynamic data associated with this binding revealed the binding to be favored by a positive change in entropy. Although molecular docking studies identified the SH3 domain as the unique binding site of these heme derivatives to erythroid spectrin, experimental results indicated a binding stoichiometry of 1 heme attached to both dimeric and tetrameric spectrin, indicating the common self-associating domain to be the unique binding site. We also noticed heme-induced structural changes in the membrane skeletal protein. Erythroid spectrin could thus act as a potential acceptor of heme, particularly relevant under disease conditions.