Studies on the stability of oxyhemoglobin A and its constituent chains and their derivatives.

Abstract Oxyhemoglobin either spontaneously or under the influence of external oxidants is converted to the high spin ferric protein (acid methemoglobin) which can be studied both optically and with electron paramagnetic resonance (EPR). The rate of ferricyanide oxidation is comparable for oxyhemoglobin A, oxyhemoglobin H (β4), and oxy α chains. p-Mercuribenzoate (PMB) derivatives of oxy α and oxy β chains react faster with this reagent than do the nonderivatized proteins. With time, the high spin form of each hemoglobin preparation is converted to various low spin forms, heretofore collectively called "hemichrome." Hemichromes are low spin derivatives of ferrihemoglobin, brought about through discrete reversible and irreversible changes of protein conformation so that atoms endogenous to the protein are now bound as the sixth ligand of the heme iron. The rate of formation of the first hemichromes from the obligatory high spin ferric protein is very slow with ferrihemoglobin A, faster with ferrihemoglobin H, and faster still with ferric α chains. This first hemichrome can be reversibly converted to deoxyhemoglobin by reduction to the hemochrome followed by anaerobic dialysis, or it can be converted quickly to oxyhemoglobin by reacting the hemochrome with CO which is then photolyzed in the presence of O2. It has been demonstrated that ferrihemoglobin cyanide prepared from high spin ferrihemoglobin H differs in structure from ferric hemoglobin cyanide prepared from the hemichrome of hemoglobin H, although the optical and EPR spectra of the two compounds are identical. With time, or under the influence of protein denaturants such as urea or salicylate, the hemichrome that can be reconstituted to oxyhemoglobin is converted to different hemichromes which are nonrenaturable to the oxy protein. Hemin added to whole globin yields high spin ferrihemoglobin A, whereas hemin added to globin prepared either from α or β subunits yields hemichrome.