Conformational studies of various hemoglobins by natural-abundance 13C NMR spectroscopy

Studies of variously liganded hemoglobins (both from human and rabbit) by natural-abundance 13C NMR spectroscopy have revealed apparent conformational differences that have been interpreted on the basis of two quaternary structures for the α2ß2 tetramer, and variable tertiary structures for the individual α and ß subunits. In solution, rabbit hemoglobins appear to have somewhat more flexibility than human hemoglobins

Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica)

We demonstrate that 5,5′-dithiobis(2-nitrobenzoate) – DTNB – reacts with only CysF9[93]β and CysB5[23]β among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica). Kequ, the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH≈5.6 and pH≈9: from a mean of 7±1 to a mean of 0.016±0.003. Quantitative analyses of the Kequ data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of Krt, the equilibrium constant for the r←→t tertiary structure transition. Krt differs significantly between the two hemoglobins: 0.744±0.04 for the major, 0.401±0.01 for the minor hemoglobin. The mean pKas of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins

Reversible reaction of 5,5V-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Acetylation of NH3 + terminal group of the h chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form

We demonstrate kinetically that the reaction of 5,5V-dithiobis(2-nitrobenzoate) with the CysF9[93]h sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3 + terminal group of GlyNA1[1]h is free, kf, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH3 + terminal group of SerNA1[1]h is acetylated, and the pH dependence profile of kf is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]h. Quantitative analyses of the complex profiles enabled us to estimate pKas of 7.47T0.3; 6.53T0.03 and 8.49T0.3 for GlyNA1[1]h, HisH21[143]h and other histidines within 2 nm of the sulfhydryl, and CysF9[93]h, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33T0.17 and 8.54T0.5 for HisH21[143]h and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]h of the minor hemoglobin, respectively

Evidence for multiple structural genes for the γ chain of human fetal hemoglobin

A sequence with a specific residue at each position was proposed for the γ chain of human fetal hemoglobin by Schroeder et al. (1) after a study in which hemoglobin from a number of individual infants was used. We have now examined in part the fetal hemoglobin components of 17 additional infants and have observed that position 136 of the γ chain may be occupied not only by a glycyl residue, as previously reported, but also by an alanyl residue

Tertiary conformational transition in sheep hemoglobins induced by reaction with 5,5´-dithiobis(2-nitrobenzoate) and by binding of inositol hexakisphosphate

We have determined the second-order reverse rate constant, kR, for the reaction of 5,5´-dithiobis(2- nitrobenzoate) – DTNB – with sheep hemoglobins as a function of pH from values of the second-order forward rate constant, kF, and the equilibrium constant, Kequ, at 25 °C: kR¼ kF Kequ. We demonstrate that (i) inositol hexakisphosphate (inositol-P6) decreases kF and kR by increasing Krt, the r⇌t tertiary conformation transition constant; (ii) the conformation favored for both the forward and reverse reactions is the r conformation. For stripped hemoglobin we obtain from the kF data a t isomer population of 34.6% (±14) prior to reaction with DTNB; from the kR data we calculate a t isomer population of 44.8% (±4) following reaction with DTNB. In the presence of inositol-P6 the latter value is increased to 79.5% (±2). These results demonstrate that an allosteric transition occurs on reaction with DTNB and on inositol-P6 binding

Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat: Variation of the equilibrium and reverse rate constants with pH

We have determined for the first time the equilibrium constant, Keq, for the reaction of Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate), with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.0 Kequ varies over four orders of magnitude — between ca 10 and 10−3 — for all hemoglobin derivatives. Using these Kequ values and published data on the dependence of the apparent second order forward rate constant, kf, on pH we have calculated the apparent second order reverse rate constant, kr, as a function of pH. This parameter increases strongly with pH, particularly above pH 7.5. Quantitative analyses of the pH dependence profiles of log10kr indicate that the reverse reaction is coupled to the ionization of two groups on the protein with pKas of 7.2±0.2 and 9.4±0.1 in the major hemoglobin and 6.7±0.3 and 8.4±0.1 in the minor hemoglobin. © 2006 Elsevier B.V. All rights reserved

Transition of hemoglobin between two tertiary conformations: Determination of equilibrium and thermodynamic parameters from the reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl group

The equilibrium constant of the reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl group of hemoglobin decreases by 2 to 3 orders of magnitude between pH 5.6 and 9. The reaction is coupled to the ionizations of two groups on the protein. At 25 °C one group has a pKa of 5.31±0.2 when hemoglobin is in its (tertiary) r conformation, typified by the thiolate anion form of CysF9[93]β; this changes to 7.73±0.4 in the (tertiary) t conformation, typified by the mixed disulfide form of the sulfhydryl. The second group ionizes with a pKa of 7.11±0.4 in the r conformation; this changes to 8.38±0.2 in the t conformation. Krt, the equilibrium constant for the r←→t isomerization process, is 0.22±0.06. The standard enthalpy and entropy changes for the isomerization are ΔHo rt=24.2 kJ mol−1 and ΔSo rt=68.8 JK−1mol−1, respectively

The primary structure of three hemoglobin chains from the indigo snake (Drymarchon corais erebennus, Serpentes): First evidence for αD chains and two β chain types in snakes

The hemoglobin of the indigo snake (Drymarchon corais erebennus, Colubrinae) consists of two components, HbA and HbD, in the ratio of 1:1. They differ in both their alpha and beta chains. The amino acid sequences of both alpha chains (alpha(A) and alpha(D)) and one beta chain (betaI) were determined. The presence of an alpha(D)chain in a snake hemoglobin is described for the first time. A comparison of all snake beta chain sequences revealed the existence of two paralogous beta chain types in snakes as well, which are designated as betaI and betaII type. For the discussion of the physiological properties of Drymarchon hemoglobin, the sequences were compared with those of the human alpha and beta chains and those of the closely related water snake Liophis miliaris where functional data are available. Among the heme contacts, the substitution alpha(D)58(E7)His-->Gln is unusual but most likely without any effect. The residues responsible for the main part of the Bohr effect are the same as in mammalian hemoglobins. In each of the three globin chains only two residues at positions involved in the alpha1/beta2 interface contacts, most important for the stability and the properties of the hemoglobin molecule, are substituted with regard to human hemoglobin. On the contrary, nine, eleven, and six alpha1/beta1 contact residues are replaced in the alpha(A), alpha(D), betaI chains, respectively

Hemoglobin Subunit-Subunit Affinity-Determinant of Hemoglobin Formation

Hemoglobin A₂ is often elevated in β-thalassemia and decreased in α-thalassemia. This might be due to hemoglobin subunit-subunit affinity variation. It has been inferred from the study of abnormal hemoglobins that the a subunits have higher affinity for β subunits than for δ subunits. However, only in one study has the affinity of α, β, and δ subunits for each other been measured. In this work we have attempted to measure the hemoglobin subunit-subunit affinity with somewhat different approach, i.e., hybridization of hemoglobin A and A₂. It is shown that hybridization and recombination of equal amounts of hemoglobins A and A₂ lead always to the formation of more hemoglobin A than A₂. Incubation of pure α, β, and δ subunits forms more hemoglobin A than A₂ as the availability of a subunits declines. It is concluded that hemoglobin a subunits have approximately four-fold higher affinity for β subunits than for the δ subunits under these experimental conditions. This subunit-subunit affinity difference, which has been attributed to the variation in molecular electrostatic charges, explains the variation of hemoglobin A₂ levels in thalassemia syndromes