Tertiary and quaternary chloride effects of the partially ligated (CN-met) hemoglobin intermediates.

Heterotropic effects of NaCl were studied using CN-met hemoglobin, which has been found to follow the same rules of homotropic cooperativity as CO-Hb and O2-Hb [Huang and Ackers, Biochemistry, 35 (1996) 704; Huang et al., Biophys. J., 71 (1996) 2094]. Modulation of heme site cooperativity by NaCl was determined in this study for all partially ligated CN-met intermediates by measuring their dimer-to-tetramer assembly free energies as a function of NaCl concentration (0.08-1.4 M; pH 7.4, T = 21.5 degrees C). Thermodynamic linkage analysis yielded the contributions to heme site binding cooperativity for all 16 reactions of the binding cascade, and also their apparent changes in bound salt. The principal findings were as follows: (i) At each [NaCl] the ten tetrameric species exhibited three discrete cooperative free energy levels; (ii) positional isomers of the doubly ligated tetramers were distributed among two of these levels according to their specific configurations of ligated sites, in conformity with the symmetry rule mechanism of hemoglobin cooperativity [Ackers et al., Science 255 (1992) 54]; (iii) the apparent moles of NaCl release followed the same configuration-specific distribution as that of heme site cooperativity, i.e., this parameter was synchronized according to the same response clusters. The system thus manifests both a "tertiary chloride effect" and a "quaternary chloride effect", which parallel the tertiary and quaternary Bohr effects [Daugherty et al., Biochemistry, 33 (1994) 10345; Perrella et al., Biochemistry, 33 (1994) 10358] and the tertiary and quaternary enthalpy effects [Huang and Ackers, Biochemistry, 34 (1995) 6316]. Comparison with findings on the stoichiometric O2-binding linkages over an identical range of conditions [Doyle et al., Biophys. Chem., 64 (1997)] revealed that the overall NaCl release upon ligating all four hemes is identical for O2 and CN-met, whereas the detailed distributions of apparent chloride release showed variations between the two ligands, i.e., CN-met Hb showed only a negligible quaternary enhancement at all [NaCl] conditions and a larger tertiary chloride effect compared with O2-Hb. Possible origins of these variations are considered.

Heterotropic effects of chloride on the ligation microstates of hemoglobin at constant water activity.

Dimer-tetramer assembly reactions of the 10 CN-met ligation microstates of hemoglobin (Hb) were analyzed as a function of NaCl concentration while maintaining constant water activity by the addition of compensating sucrose. The assembly free energy for fully ligated cyanomet Hb and for fully oxygenated Hb becomes less favorable by 1.8 kcal when [NaCl] is increased from 0.08 to 0.7 M, whereas that of unligated Hb is practically insensitive to changes in [NaCl]. Values of 1.6 and 0.3 mol chloride release were found for the assembly of fully ligated and deoxy Hb, respectively; i.e., a net release of 1.3 mol chloride is coupled to the ligation of tetramers for both oxygen and cyanomet ligation. The ligation-linked salt component at constant water activity was evaluated to be 1.0 mol for the full oxygenation of the Hb tetramer in agreement with the overall value previously reported. When the detailed salt linkages accompanying all 16 stepwise cyanomet ligation reactions were experimentally resolved, only two "chloride" effects were found. The first chloride effect correlates with the ligation steps, which create tertiary constraint, and the second effect is coupled to the six switchpoints of quaternary T-->R transition. The distribution of these chloride effects agrees closely with predictions of the "symmetry rule mechanism." The total chloride release for CN-met ligation is in good agreement with that for oxygenation. Free energy contributions to assembly and cooperativity arising from the osmotic effects of chloride were found to be small for all ligation species.