Understanding mechanisms in cooperative proteins requires the analysis of the intermediate ligation states. The release of hydrogen ions at the intermediate states of native and chemically modified hemoglobin, known as the Bohr effect, is an indicator of the protein tertiary/ quaternary transitions, useful for testing models of cooperativity. The Bohr effects due to ligation of one subunit of a dimer and two subunits across the dimer interface are not additive. The reductions of the Bohr effect

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... due to the chemical modification of a Bohr group of one and two ␣ or ␤ subunits are additive. The Bohr effects of monoliganded chemically modified hemoglobins indicate the additivity of the effects of ligation and chemical modification with the possible exception of ligation and chemical modification of the ␣ subunits. These observations suggest that ligation of a subunit brings about a tertiary structure change of hemoglobin in the T quaternary structure, which breaks some salt bridges, releases hydrogen ions, and is signaled across the dimer interface in such a way that ligation of a second subunit in the adjacent dimer promotes the switch from the T to the R quaternary structure. The rupture of the salt bridges per se does not drive the transition. A vast amount of data on the structure/function of human hemoglobin in solution apparently supports the mechanism of a concerted transition between two quaternary structural states in the course of ligand binding, in agreement with the Monod-Wyman-Changeaux model (1). Due to cooperativity, the end states largely prevail on species in a partial state of ligation under equilibrium conditions, masking the functional properties of the intermediate species. This is demonstrated by the close agreement between the isotherms of CO binding calculated from the experimental distributions of the CO ligation intermediates according to the Monod-Wyman-Changeaux model and the alternative Koshland-Nemethy-Filmer model, which assumes transitions through intermediate structural/ functional states (2), as shown by Perrella and Di Cera (3). The functional/structural studies of the intermediates still provide the most critical test for any model of cooperativity. Such studies are difficult because of the high rates of the dissociation and association reactions of the physiological ligand, the com-plexity of the intermediate ligation states Fig. 1 , and the instability of tetrameric hemoglobin. The partially liganded hemoglobin tetramers reversibly dissociate into dimers faster than the rate of resolution of the separation techniques (4), and dimer rearrangement reactions occur under nonequilibrium conditions, as depicted in Fig. 2 . In a previous study of the Bohr effect of the intermediate ligation states (5) , the problem of the ligand mobility was circumvented by using cyanide bound to the ferric subunits to mimic ligation and a cryogenic technique to determine the proportion of any asymmetrical hybrid species in equilibrium with the respective symmetrical parental species (6). This information was needed to calculate the contribution of each species from the total Bohr effect of a mixture of hybrid and parental species. The study of the pH dependence of the Bohr effects of the mono-and diliganded intermediates revealed the absence of additivity of the effects, an important clue to the mechanism of tertiary/quaternary transitions in ligand binding to hemoglobin. However, the discovery by Shibayama et al. (7) that the cyanomet intermediates undergo valency exchange has made such studies questionable. We have now repeated the measurement of the Bohr effect of the mono-and some diliganded species under conditions of slight or negligible valency exchange, confirming the results of the previous study. Using the same technical approach we have measured the decrease in Bohr hydrogen ions in hemoglobin derivatives in which either one or both Bohr groups of the ␣ and ␤ subunits of deoxy hemoglobin and of the deoxy/cyanomet intermediates were chemically modified by carbamoylation (8) and by the NEM 1 reaction of cysteine F␤93 (9). We found that the functional effects of the single and double chemical modifications were additive, as were the combined effects of ligation and chemical modification, with just one possible exception. These findings help define the role of the salt bridges with regard to the stabilization of the hemoglobin T quaternary structure, which was described by Perutz in his stereochemical mechanism of cooperativity (10). MATERIALS AND METHODS Hemoglobin Purification-HbA 0 was obtained from normal adult blood and HbS from heterozygous donors. The hemoglobins were purified by ion exchange chromatography on CM-52 cellulose, as previously described (5), equilibrated with 0.2 M KCl, and stored in liquid nitrogen at a concentration of 6 mM in heme. Preparation of NES Hemoglobin-Samples (4.5 g) of HbO 2 were reacted with a 5-fold excess of NEM at 4°C and pH 7.3 for 2 h (11). The reactants were gel-filtered on Sephadex G-25 equilibrated with 5 mM potassium phosphate, 0.5 mM Na 2 EDTA, pH 6.8, and loaded onto a column (8 ϫ 27 cm) of CM-52-cellulose equilibrated with the same