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 st udy of the structure/function relationships in proteins. One of the most di fficult, and so far, unattained objectives of hemoglobin research has been the study of the hemoglobin molecules in a state of partial ligation with o xygen, or intermediates, as a means of testing theories of cooperativity. A cryogenic technique has been developed for the isolation, identification a nd 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 approach es 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 solu tion. (C) 1999 Elsevier Science B.V. All rights reserved.