Quantitative analysis of the association of human hemoglobin with the cytoplasmic fragment of band 3 protein.
The association of the isolated cytoplasmic fragment of band 3 protein with human hemoglobin was studied by rate zonal centrifugation in sucrose density gradients, by quenching of fragment fluorescence by hemoglobin, and by flash photolysis of carbon monoxidebound hemoglobin as a function of fragment concentration. The centrifugation results showed that both proteins interact and that the interaction is abolished upon addition of glyceraldehyde-3-phosphate dehydrogenase. The fractions eluted from the density gradient were analyzed further by spectrophotometric and gel electrophoretic methods. Two types of complexes could be identified, one containing the equivalent of 1 hemoglobin tetramer/dimer of cytoplasmic fragment and another containing 2 tetramers of hemoglobin/dimer of fragment. Flash photolysis and fluorescence-quenching experiments showed that liganded hemoglobin is stabilized as the alpha beta dimer when bound to the fragment, a result almost identical with that seen for membranebound hemoglobin in previous studies. The results further suggest that there are two binding sites for the alpha beta dimer of hemoglobin on one monomer of the cytoplasmic fragment but only one mutually exclusive hemoglobin tetramer binding site, suggesting the possibility of conformational isomerism when the fragment with two dimers bound isomerises to a fragment monomer with one hemoglobin tetramer bound. Finally, despite the stabilization of the dimeric state of liganded hemoglobin when bound to the fragment, estimates of the hemoglobin dimer and tetramer binding constants suggest that the hemoglobin tetramer binds more tightly by about 2 orders of magnitude.
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins / Q000378:metabolism*; D002501:Centrifugation, Zonal; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010108:Oxyhemoglobins / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D006454:Hemoglobins; D010108:Oxyhemoglobins