Reconstitution ofnative humanhemoglobin fromseparated globin chains andalloplex intermediates

Acomplete experimental format isgiven for thereconstitution ofhumanhemoglobin fromtheseparated heme-free a-and-globin chains (a', ft') andhemin, bytwo alternative routes. Based ontheir oxygen binding properties, thereaction oftheferri-forms with reducing agent, andthere- sponse oftheoxygen binding curves topHvariation andtothe addition ofthealosteric effector 2,3-diphosphoglycerate, the molecules arenative. Onereconstitution route uses direct ad- dition ofhemin totheseparated globin chains with production oftheseparated subunits, which canthen berecombined and reduced. This procedure occasions losses byprecipitation inthe heme-addition step except athigdilutions, andtheyields are low. Inthesecond pathway, either globin chain ismixed with thecomplementary untreated subunit toformthehalf-filled (with heme) intermediates, which combine stoichiometrically with hemin. Noprecipitation accompanies these reactions. For a-globin, theyield isabout 50%because ofincomplete combi- nation with theheme-containing Pchain. For-globin, theyield isbetter than 70%.Itissuggested that experiments intended totest either globin chain should usethesecond route inprep- aration forstructural orfunctional comparisons withnative hemoglobin.