Understanding mechanisms in a cooperative protein: the CO ligation intermediates of hemoglobin

Hemoglobin is a regulatory component of the oxygen transport to the tissues, and for decades has been a prototype to develop new strategies for the study of the structure/function relationships in proteins. One of the most difficult, and so far, unattained objectives of hemoglobin research has been the study of the hemoglobin molecules in a state of partial ligation with oxygen, or intermediates, as a means of testing theories of cooperativity. A cryogenic technique has been developed for the isolation, identification and quantification of the reaction intermediates of hemoglobin and CO, which in many aspects is a close approximation to the physiological ligand. The technical features that are crucial for the evaluation of the significance of the experimental data obtained using this technique and various approaches to the analysis of the data are reported. The discussion points out the importance of accessing direct information on the nature and concentrations of the intermediates in solution to clarify mechanisms of cooperativity as opposed to the less informative studies of the bulk properties of the solution.