Hemoglobin Binds to the Amino-Terminal 23-Residue Fragment of Human Erythrocyte Band 3 Protein

Abstract

Hb, aldolase and glyceraldehyde 3-phosphate dehydrogenase are known to bind to the cytoplasmic domain of band 3 protein. Binding of glycolytic enzymes to band 3 protein is inhibited by its amino-terminal fragments. To precisely localize the sequence portion of band 3 protein to which Hb binds, and to see whether the same region of amino-acid sequence binds both Hb and glycolytic enzymes, a simple, direct solid-phase binding assay was developed. Peptides generated from the 23-kDa [kilodalton] fragment by trypsin, cyanogen bromide and mild and hydrolysis were used as inhibitors to determine the minimal sequence structure involved in the binding of the 23-kDa fragment to Hb. The shortest peptide which inhibits the binding of the 23-kDa fragment is an acid cleavage peptide containing the sequence positions 1-23. This sequence is unusual as 14 of its residues are negatively charged; it contains no basic residues and has its amino terminus blocked. Using aldolase, glyceraldehyde-3-phosphate dehydrogenase and Hb as competitive inhibitors in the binding of 23-kDa fragment, the affinity of Hb to this fragment appears several-fold weaker than that of both the enzymes. Glycolytic enzymes and Hb bind competitively to the same polyanionic sequence region of band 3 protein.