Hemoglobin binds to the amino-terminal 23-residue fragment of human erythrocyte band 3 protein.
Hemoglobin, 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 hemoglobin binds and to see whether the same region of amino-acid sequence binds both hemoglobin and glycolytic enzymes, a simple, direct solid-phase binding assay was developed. Peptides generated from the 23-kDa fragment by trypsin, cyanogen bromide and mild acid hydrolysis were used as inhibitors to determine the minimal sequence structure involved in the binding of the 23-kDa fragment to hemoglobin. The shortest peptide which inhibits the binding of the 23-kDa fragment is an acid cleavage peptide containing the sequence positions 1 to 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 hemoglobin as competitive inhibitors in the binding of 23-kDa fragment, the affinity of hemoglobin to this fragment appears several-fold weaker than that of both the enzymes. These findings demonstrate that glycolytic enzymes and hemoglobin bind competitively to the same polyanionic sequence region of band 3 protein.
D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001665:Binding Sites; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006441:Hemoglobin A / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000097:blood; D011485:Protein Binding
D001457:Anion Exchange Protein 1, Erythrocyte; D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D010446:Peptide Fragments; D006441:Hemoglobin A; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005634:Fructose-Bisphosphate Aldolase