Interaction of hemoglobin and its component alpha and beta chains with band 3 protein.
The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)
D019794:2,3-Diphosphoglycerate; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding
D001457:Anion Exchange Protein 1, Erythrocyte; D004163:Diphosphoglyceric Acids; D006454:Hemoglobins; D046911:Macromolecular Substances; D019794:2,3-Diphosphoglycerate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate