Reactivity difference between haemoglobins. Part XIX

Abstract

The formation constants of rhe azide complex of two methaemoglobins with widely different pH_ch values, guinea pig and pigeon methaemoglobin, have been measured as a function of ionic strength. These results, combined with previous results for human methaemoglobin A and C, confirm that the ionic-strength dependence of the formation constant can be quantitatitiely accounted for in terms of a dielectric cavity model for the protein except over narrow range of pH in the region of the characteristic pH for each methaemoglobin. This breakdown of the model interpreted in terms of a pH-dependent configurational change involving charged groups on the surface of the molecule.

The formation constants of the azide complex of the haem groups attached to the α and β polypeptide chains within the human methaemoglobin tetramer have been shown to differ by a factor of 5. It is shown that this difference can quantitatively account for the value of the Hill constant of 0.91 ± 0.01 obtained when measurements made at 405 nm, a wavelength which indicates reaction at either the α or β haems. It is concluded that there positeve or negative co-operativity between the haem groups when methaemoglobin reacts with azide ion.

The formation constants for the reaction of several methaemoglobins and sperm whale metmyoglobin formate ion have been measured as a function of pH and temperature at I= 0.25M. Formate ion as resembles fluoride ion in that all the haemoglobin species have closely similar affinities for the ligand, confirming differences between species in the affinity for a particular ligand is attributable to the configuration which accompanies the spin-state change. The pH dependence of ΔH° resembles that for azide and cyanide showing that factors other than the spin-state change determine the type of ΔH° behaviour shown by a Iigand.

The apparent paradox that the large pH variations in the enthalpy of formation of methaemoglobin complexes are accompanied by large temperature variations of the number of protons released upon ligand binding, as would be required by the Wyman relationship, is re-examined. The Wyman relationship is derived by an alternative procedure, a tacit assumption is made explicit. It is shown that the large pH variation in ΔH° can arise if the configuration charged groups on the surface of the molecule is different in methaemoglobin and its complex. When such a difference in configuration exists the addition of the same number of protons to methaemoglobin and its complex give rise to different changes in the partial molar entropy of the two species.

The formation constant for the azide complex of glycera methaemoglobin has been determined as a function and temperature. The affinity of this haemoglobin for azide ion is much lower than thatfora typical mammalian methaemoglobin. This difference is attributed to the close proximity to the iron atom of an aspartic acid residue postion E5(57) in glycera methaemoglobin. The pH variation of the enthalpy of complex formation is similar to observed for a typical mammalian methaemoglobin in spite of the absence of the distal imidazole group in glycera methaemoglobin. This is accounted for in terms of the proximity to the iron atom of the E5(57) aspartic acid residue.

The formation constants of the azide complex of the isolated polypeptide chains of human and dog methaemoglobins have been determined as a function of pH and temperature. The variation of the enthalpy of complex formation resembles that of a typical methaemoglobin, the characteristic pH for the α chains of human methaemoglobin and the β chains of human and dog methaemoglobin falling on the previously noted correlation line between pH_ch and a function of the charged amino-acid in the molecule. The deviation of the α chain of dog methaemoglobin from the correlation line is discussed.

The formation constants of the azide complex of the valency hybrids of human haemoglobin and the hybrids of dog and human methaemoglobin have been determined as a function of pH and temperature. The valency hybrids of human haemoglobin have the same characteristic pH as human methaemoglobin showing that pH_ch is a property of the haemoglobin tetramer even if only one pair of haems is reacting with ligand. The dog–human hybrid met-haemoglobins show the typical pH variation of the enthalpy of complex formation, there being a characteristic pH for the tetrameric species which can be calculated from the characteristic pH values of the isolated chain. That a tetra-merit methaemoglobin shows a single characteristic pH rather than two, corresponding to those of the isolated α and β polypeptide chains, is accounted for by structural constraints imposed by one haem on the other leading to a con-certed transition -from the acid to the alkaline configuration at the characteristic pH.

The formation constants for the reaction of azide, formate, and fluoride ions with various methaemoglobins chemically modified in differentways have been determined as a function of pH and temperature. Chemical modification has two distinct effects on the pl l variation of the enthalpy of complex formation. (i) The characteristic pH of a modified methaemoglobin shifts towards that of the unmodified polypeptide chain. This is explained in terms of a reduction in the constraint imposed by the modified chain on the unmodified chain, such that the pH at which the concerted configurational change occurs (pH_ch) is determined primarily by the unmodified chain, (ii) The form of the variation may change, the extreme case being where the customary maximum in the –ΔH° against pH plot becomes a minimum for methaemoglobin modified with iodoacetamide reacting with formate ion. This variable behaviour is interpreted in terms of an equilibrium between the ‘ hydrogen in ’ and ‘ hydrogen out ’ configuration on the distal imidazole group.

The magnitude of the Bohr effect for a number of chemically modified and hybrid haemoglobin species has been determined as a function of pH. The dog–human hybrid haemoglobins fit the previously noted correlation between the magnitude of the acid Bohr effect, Δh⁺_5,3, and the characteristic pH of a haemoglobin, implying that no special explanation is required for the very small acid Bohr effect of α^Aβ₂^dog. The Δh⁺_5,3 a for the vacancy hybrid human haemoglobin, α⁺β₂(O₂)₂, falls on the correlation with pH_ch but that for the hybrid α₂(O₂)₂β₂⁺ does not. Δh⁺_5,3 Values for human methaemoglobins enzymatically digested with carboxypeptides A and B fall on the correlation line. A mechanism is proposed to account for the correlation between Δh⁺_5,3 and pH_ch.