Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.
A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free alpha- and beta-globin chains (alpha degrees, beta degrees) and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the allosteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at high dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For alpha-globin, the yield is about 50% because of incomplete combination with the heme-containing beta chain. For beta-globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.
D000495:Allosteric Site; D004163:Diphosphoglyceric Acids / Q000097:blood; D005914:Globins; D006418:Heme / Q000031:analogs & derivatives; D006427:Hemin; D006441:Hemoglobin A / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008773:Methylphenazonium Methosulfate; D009243:NAD; D010084:Oxidation-Reduction; D010100:Oxygen / Q000097:blood; D010108:Oxyhemoglobins / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D004163:Diphosphoglyceric Acids; D006454:Hemoglobins; D010108:Oxyhemoglobins; D009243:NAD; D008773:Methylphenazonium Methosulfate; D006418:Heme; D006427:Hemin; D005914:Globins; D006441:Hemoglobin A; D010100:Oxygen