Ligand Binding Kinetic Studies on the Hybrid Hemoglobin α(Carp):β(Human)

A Hemoglobin with a Restricted Allosteric Range

Dixie J. Goss, Lawrence J Parkhurst

Research output: Contribution to journalArticle

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Abstract

The preparation of native α- and β-chains of carp hemoglobin and the preparation of the hybrid hemoglobin α(carp):β(human), hybrid I, are described. CO association and dissociation kinetics were determined for the hybrid hemoglobin α(carp):β(human) as a function of fractional saturation by stopped-flow and by flash and laser photolysis. Oxygen dissociation from the liganded distribution of con-formers 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.

Original languageEnglish (US)
Pages (from-to)2174-2179
Number of pages6
JournalBiochemistry
Volume23
Issue number10
DOIs
StatePublished - Jan 1 1984

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Carbon Monoxide
Hemoglobins
Carps
Ligands
Kinetics
Association reactions
Oxygen
Photolysis
Rate constants
Lasers
Equilibrium constants
Flow measurement
Theophylline
Free energy
hybrid hemoglobins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ligand Binding Kinetic Studies on the Hybrid Hemoglobin α(Carp):β(Human) : A Hemoglobin with a Restricted Allosteric Range. / Goss, Dixie J.; Parkhurst, Lawrence J.

In: Biochemistry, Vol. 23, No. 10, 01.01.1984, p. 2174-2179.

Research output: Contribution to journalArticle

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abstract = "The preparation of native α- and β-chains of carp hemoglobin and the preparation of the hybrid hemoglobin α(carp):β(human), hybrid I, are described. CO association and dissociation kinetics were determined for the hybrid hemoglobin α(carp):β(human) as a function of fractional saturation by stopped-flow and by flash and laser photolysis. Oxygen dissociation from the liganded distribution of con-formers 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.",
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