title: Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane.
abstract: The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.
mesh_terms: D000818:Animals; D001665:Binding Sites; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006021:Glycophorins / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000097:blood; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000097:blood; D011817:Rabbits; D010738:Type C Phospholipases
chemical_list: D006021:Glycophorins; D006454:Hemoglobins; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010743:Phospholipids; D010738:Type C Phospholipases