Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.

Abstract

A complete experimental format is given for the reconstitution of human Hb from the separated heme-free .alpha.- and .beta.-globin chains (.alpha..degree., .beta..degree.) and hemin, by 2 alternative routes. Based on their O2 binding properties, the reaction of the ferri-forms with reducing agent, and the response of the O2 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 2nd 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%. Experiments intended to test either globin chain should probably use the 2nd route in preparation for structural or functional comparisons with native Hb.