Binding Reaction of Hemin to Globin

Abstract

Binding of hemin to globin was studied in the presence of 25 mM caffeine by measuring CD and optical absorption changes in the Soret region. CD and optical absorption spectra after mixing equimolar amounts of hemin and globin were the same as those of ferric hemoglobin. In contrast, addition of excess globin to hemin formed a complex that was distinguishable from ferric hemoglobin in terms of the CD and optical absorption spectra. By comparing the spectra of the complex with those of various hemoglobin derivatives, it was concluded that the complex was globin which carried a hemin exclusively on the α chain. This means that the α chain of the globin molecule has a greater affinity for hemin than the β chain, as observed by other investigators using hemin-cyanide. The rate of binding of hemin to globin was estimated by the use of CD and optical absorption stopped-flow apparatus. The rate of hemin binding to the α chain of globin was obtained by mixing hemin and excess globin, and that to the β chain was obtained by mixing equimolar concentrations of hemin and globin. The results showed that (1) hemin was bound to the a chain in the globin molecule to form a transient intermediate, followed by its transformation into another inter mediate, (2) the transformation was the rate-limiting step, and (3) the β chain in the globin molecule had a greater affinity for hemin after hemin binding to the a chain than before.