Functional heterogeneity of the alpha and beta subunits in the association reaction between hemoglobin and carbon monoxide

A technique is described for the rapid inactivation and removal of excess ferricyanide used for the non-cryogenic oxidation of the unliganded subunits of the intermediates in the association reaction between hemoglobin and carbon monoxide. Under these conditions the asymmetric oxidized intermediates, which dissociate into non-identical dimers, disproportionate into their parent tetramers and four species, Hb+, HbCO, alpha 2+ beta 2CO, alpha 2CO beta 2+, are isolated by non-cryogenic isoelectric focusing. The relative concentrations of species alpha 2CO beta 2+ and alpha 2+ beta 2CO measure the overall distribution of the ligand between the alpha and beta subunits in the association reaction. At 20 degrees C in 0.1 M KCl, pH 7, preferential CO binding to the beta subunits was observed, in agreement with observations made by the cryogenic technique for the isolation of the intermediates [M. Perrella, N. Davids and L. Rossi-Bernardi, J. Biol. Chem. 267 (1992) 8744].