THE INTERMEDIATE COMPOUNDS BETWEEN HUMAN-HEMOGLOBIN 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.