Ligand binding kinetic studies on the hybrid hemoglobin alpha(carp):beta(human): a hemoglobin with a restricted allosteric range.
The preparation of native alpha- and beta-chains of carp hemoglobin and the preparation of the hybrid hemoglobin alpha(carp):beta(human), hybrid I, are described. CO association and dissociation kinetics were determined for the hybrid hemoglobin alpha(carp):beta(human) as a function of fractional saturation by stopped-flow and by flash and laser photolysis. Oxygen dissociation from the liganded distribution of conformers and overall oxygen association were studied by laser photolysis. An allosteric model was used to fit successfully all of the CO and oxygen kinetics and equilibria for this hybrid. In this model, it was assumed that the conformational changes were rapid with respect to ligand binding. A linear free-energy relationship relating rate constants and equilibrium constants was assumed in order to reduce the number of fitting parameters. In the allosteric model itself, it was assumed that L, but not c, varied with pH. For CO, the allosteric parameter c was 0.24; for oxygen, the value was 0.14. At pH 7, L was 80. In hybrid I, the calculated CO association rate constants for the R and T states differ by only a factor of 2. From the equilibrium data, with no assumptions as to the rate of conformational equilibrations , one can show that the allosteric model requires that the measured CO association constant from stopped-flow measurements be assigned to the T state. This state, however, is poised approximately midway between a carp-human T state and a carp hemoglobin R state. In this hybrid, neither chain shows normal R-state behavior; rather, the liganded state is also intermediate between R and T. In this hemoglobin, the low cooperativity appears to be associated with a very restricted range for the R-T conformational change.
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D005399:Fishes; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D010100:Oxygen / Q000378:metabolism; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins
D006454:Hemoglobins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C041823:hybrid hemoglobins; D002248:Carbon Monoxide; D010100:Oxygen