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The Self-Association and Oxygen Equilibrium of Hemoglobin from the Lamprey, Entosphenus japonicus

From the ¹ From the Department of Biochemistry, Kanazawa University School of Medicine, Kanazawa, Japan

Quantitative studies were made on the association of hemoglobin from the lamprey, Entosphenus japonicus, in a wide range of concentrations (0.01 to 3.4 mm) and pH values (5.6 to 8.0) with the aids of sedimentation equilibrium, sedimentation velocity, and diffusion measurements. Oxygen equilibrium was determined also in a wide range of concentrations by colorimetry and manometry.

Molecular weights of the monomer and the largest aggregate were calculated to be 17,300 and 67,300, respectively, which correspond to that of the subunit of human hemoglobin and that of tetrameric human hemoglobin, respectively. The data of the sedimentation velocity and sedimentation equilibrium were computer analyzed assuming the rapid equilibrium of the monomer-dimer-tetramer, and the association constants for both deoxygenated and oxygenated lamprey hemoglobin were obtained. The association-dissociation equilibrium was dependent on pH. For the deoxygenated hemoglobin, the maximum value of 8.1 x 10⁴ m^-1 for K₂, the constant for the monomer-dimer equilibrium, and 6.5 x 10³ m^-1 for K₄, the constant for the dimer-tetramer equilibrium, were obtained at pH 5.9, and the values of both association constants decreased at higher pH. The association constants for oxyhemoglobin were much smaller than those for deoxyhemoglobin. The fractions of the dimers and tetramers, calculated for the lamprey deoxygenated hemoglobin under the physiological conditions in red cells, were 10% and 85%, respectively, and those for oxyhemoglobin were 36% and 15%, respectively.

In the oxygen equilibrium of lamprey hemoglobin, it was shown that the oxygen partial pressure at half-saturation, p increased with increasing concentrations of hemoglobin, namely from 5 mm Hg at 2 µm to 350 mm Hg at 7.5 mm at pH 5.9, and that the heme-heme interaction constant, n, increased from 1.1 at very low concentration of hemoglobin to 1.6 at 0.6 mm, and then decreased to 1.0 at 7.5 mm. An assumption was made that monomers and tetramers each have a different constant affinity for oxygen which we determined experimentally from the oxygen equilibrium at a very low concentration and a very high concentration of hemoglobin, respectively. Oxygen equilibrium curves for lamprey hemoglobin were calculated from these two oxygen equilibrium constants and the association-dissociation constants determined both for deoxyhemoglobin and oxyhemoglobin. The results agreed well with those obtained experimentally, and the model was shown to be able to account for the characteristic oxygen binding properties of lamprey hemoglobin, namely heme-heme interaction, oxygen affinity, and the Bohr effect.

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