The intermediate compounds between human hemoglobnin and carbon monoxide at equilibrium and during approach to equilibrium

The procedure of Perrella et al. (Perrella, M., Benazzi, L., Cremonesi, L., Vesely, S., Viggiano, G., and Rossi-Bernardi, L. (1983) J. Biol. Chem. 258, 4511-4517) for trapping the intermediate compounds between human hemoglobin and carbon monoxide was validated by quantitatively determining during the approach to equilibrium all the species present in a solution containing large amounts of intermediates. An accurate estimate of the intermediate compounds at 50% carbon monoxide saturation in 0.1 M KCl, pH 7, at 22°C, allowed the calculation, according to Adair's scheme, of the four equilibrium constants. At 50% ligand saturation, the pool of intermediate species was about 12% of the total. A slightly greater concentration of tri-liganded than mono-liganded species was found. Carbon monoxide to β chains in slightly greater excess with respect to α chains in both the mono- and tri-liganded species. The symmetrical bi-liganded intermediates, α₂β₂(CO) and α₂(CO)β₂, were absent. The nature of the bi-liganded intermediate found to be present in detectable amounts by our technique has yet to be clarified: it could be either the asymmetrical species (αβ)(α(CO)β(CO)) and (αβ(CO)β)) or both of them. Such a finding on the functional heterogeneity among the four possible bi-liganded intermediates is consistent with hypotheses of the existence of more than two quaternary structures in the course of ligand binding to hemoglobin.