Ligand Binding Kinetic Studies on the Hybrid Hemoglobin α(Human):β(Carp): A Hemoglobin with Mixed Conformations and Sequential Conformational Changes

Lawrence J Parkhurst, Dixie J. Goss

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Abstract

Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) a(human):β(carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human α-chain is R state and the carp β-chain is T state. We have no evidence at this pH that the carp β-chain ever assumes the R conformation. At pH 6, the human α-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp β-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human α-chains are in the R state, the two β-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the β-chains first assume T-state properties, followed by the α-chains. The rate of the R-T conformational change for the carp β-chains is at least 300 times greater than that for the human α-chains. At pH 9, the R → T conformational transition rate is at least 200 times slower than that for human hemoglobin. The carp β-chain R state at this pH reacts twice as rapidly with CO as in carp hemoglobin. Diagrams are presented showing all conformations supported by kinetic evidence. The ligand kinetics in the hybrid suggest that in carp hemoglobin, the β-chains prevent the carp /3-chains from assuming full R-state properties and are themselves biased toward the T conformation. The marked tendency of the carp /3-chains to assume the T state lends strong support to the model that assigns a central role to the /3-chain in the Root effect [Perutz, M. F., & Brunori, M. (1982) Nature (London) 299, 421-426], From the data at hand, however, it does not appear possible to assign a definite number of Bohr protons to the four individual chains in carp and human hemoglobins.

Original languageEnglish (US)
Pages (from-to)2180-2186
Number of pages7
JournalBiochemistry
Volume23
Issue number10
DOIs
StatePublished - May 1984

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Carps
Conformations
Hemoglobins
Ligands
Kinetics
Carbon Monoxide
Phytic Acid
hybrid hemoglobins
Photolysis
Dimers
Protons
Oxygen
Ligation
Hand

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{391c4e801a4045e59c692311a1d25df9,
title = "Ligand Binding Kinetic Studies on the Hybrid Hemoglobin α(Human):β(Carp): A Hemoglobin with Mixed Conformations and Sequential Conformational Changes",
abstract = "Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) a(human):β(carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human α-chain is R state and the carp β-chain is T state. We have no evidence at this pH that the carp β-chain ever assumes the R conformation. At pH 6, the human α-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp β-chains, on the other hand, react 50{\%} more rapidly in the liganded conformation than in carp hemoglobin, and while the human α-chains are in the R state, the two β-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the β-chains first assume T-state properties, followed by the α-chains. The rate of the R-T conformational change for the carp β-chains is at least 300 times greater than that for the human α-chains. At pH 9, the R → T conformational transition rate is at least 200 times slower than that for human hemoglobin. The carp β-chain R state at this pH reacts twice as rapidly with CO as in carp hemoglobin. Diagrams are presented showing all conformations supported by kinetic evidence. The ligand kinetics in the hybrid suggest that in carp hemoglobin, the β-chains prevent the carp /3-chains from assuming full R-state properties and are themselves biased toward the T conformation. The marked tendency of the carp /3-chains to assume the T state lends strong support to the model that assigns a central role to the /3-chain in the Root effect [Perutz, M. F., & Brunori, M. (1982) Nature (London) 299, 421-426], From the data at hand, however, it does not appear possible to assign a definite number of Bohr protons to the four individual chains in carp and human hemoglobins.",
author = "Parkhurst, {Lawrence J} and Goss, {Dixie J.}",
year = "1984",
month = "5",
doi = "10.1021/bi00305a012",
language = "English (US)",
volume = "23",
pages = "2180--2186",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Ligand Binding Kinetic Studies on the Hybrid Hemoglobin α(Human):β(Carp)

T2 - A Hemoglobin with Mixed Conformations and Sequential Conformational Changes

AU - Parkhurst, Lawrence J

AU - Goss, Dixie J.

PY - 1984/5

Y1 - 1984/5

N2 - Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) a(human):β(carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human α-chain is R state and the carp β-chain is T state. We have no evidence at this pH that the carp β-chain ever assumes the R conformation. At pH 6, the human α-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp β-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human α-chains are in the R state, the two β-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the β-chains first assume T-state properties, followed by the α-chains. The rate of the R-T conformational change for the carp β-chains is at least 300 times greater than that for the human α-chains. At pH 9, the R → T conformational transition rate is at least 200 times slower than that for human hemoglobin. The carp β-chain R state at this pH reacts twice as rapidly with CO as in carp hemoglobin. Diagrams are presented showing all conformations supported by kinetic evidence. The ligand kinetics in the hybrid suggest that in carp hemoglobin, the β-chains prevent the carp /3-chains from assuming full R-state properties and are themselves biased toward the T conformation. The marked tendency of the carp /3-chains to assume the T state lends strong support to the model that assigns a central role to the /3-chain in the Root effect [Perutz, M. F., & Brunori, M. (1982) Nature (London) 299, 421-426], From the data at hand, however, it does not appear possible to assign a definite number of Bohr protons to the four individual chains in carp and human hemoglobins.

AB - Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) a(human):β(carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human α-chain is R state and the carp β-chain is T state. We have no evidence at this pH that the carp β-chain ever assumes the R conformation. At pH 6, the human α-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp β-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human α-chains are in the R state, the two β-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the β-chains first assume T-state properties, followed by the α-chains. The rate of the R-T conformational change for the carp β-chains is at least 300 times greater than that for the human α-chains. At pH 9, the R → T conformational transition rate is at least 200 times slower than that for human hemoglobin. The carp β-chain R state at this pH reacts twice as rapidly with CO as in carp hemoglobin. Diagrams are presented showing all conformations supported by kinetic evidence. The ligand kinetics in the hybrid suggest that in carp hemoglobin, the β-chains prevent the carp /3-chains from assuming full R-state properties and are themselves biased toward the T conformation. The marked tendency of the carp /3-chains to assume the T state lends strong support to the model that assigns a central role to the /3-chain in the Root effect [Perutz, M. F., & Brunori, M. (1982) Nature (London) 299, 421-426], From the data at hand, however, it does not appear possible to assign a definite number of Bohr protons to the four individual chains in carp and human hemoglobins.

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