Contribution of the γ-carboxyl group of glu-43(β) to the alkaline bohr effect of Hemoglobin A

A. Seetharama Acharya

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Glu-43(β) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(β) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the γ-carboxyl group of Glu-43(β) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(β) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pKa of 7.0 for the γ-carboxyl group of Glu-43(β). This pKa is considerably higher than the pKa of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pKa of the γ-carboxyl group of Glu-43(β) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography. The chemical characterization of this derivative of hemoglobin A, involving the separation of the modified β-globin by RPHPLC, isolation of the modified tryptic peptide by RPHPLC, and amino acid sequencing of the modified tryptic peptide, has established that this is a homogeneous derivative of hemoglobin in which both γ-carboxyl groups of Glu-43(β) have been amidated. The amidation of Glu-43(β) increases the O2 affinity slightly without any influence on the Hill coefficient. A comparison of the Bohr proton titration of hemoglobin A with that of the disubstituted derivative as a function of pH established the contribution of the γ-carboxyl group of Glu-43(β) to the alkaline Bohr effect of the protein. Glu-43(β) is located at the α11 interface of hemoglobin A, and the increase in the pKa of the γ-carboxyl group of Glu-43(β) upon deoxygenation of the protein apparently reflects an increase in the hydrophobicity of the α11 interface of the molecule.

Original languageEnglish (US)
Pages (from-to)7231-7236
Number of pages6
JournalBiochemistry
Volume31
Issue number32
StatePublished - 1992

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Hemoglobin A
Chemical reactivity
Proteins
Derivatives
Conformations
Carbodiimides
Peptides
Globins
Protein Sequence Analysis
Glycosylated Hemoglobin A
Nucleophiles
Hydrophobic and Hydrophilic Interactions
Cellulose
Hydrophobicity
Ligation
Chromatography
Protons
Titration
Hemoglobins
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

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Contribution of the γ-carboxyl group of glu-43(β) to the alkaline bohr effect of Hemoglobin A. / Seetharama Acharya, A.

In: Biochemistry, Vol. 31, No. 32, 1992, p. 7231-7236.

Research output: Contribution to journalArticle

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title = "Contribution of the γ-carboxyl group of glu-43(β) to the alkaline bohr effect of Hemoglobin A",
abstract = "Glu-43(β) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(β) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the γ-carboxyl group of Glu-43(β) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(β) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pKa of 7.0 for the γ-carboxyl group of Glu-43(β). This pKa is considerably higher than the pKa of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pKa of the γ-carboxyl group of Glu-43(β) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography. The chemical characterization of this derivative of hemoglobin A, involving the separation of the modified β-globin by RPHPLC, isolation of the modified tryptic peptide by RPHPLC, and amino acid sequencing of the modified tryptic peptide, has established that this is a homogeneous derivative of hemoglobin in which both γ-carboxyl groups of Glu-43(β) have been amidated. The amidation of Glu-43(β) increases the O2 affinity slightly without any influence on the Hill coefficient. A comparison of the Bohr proton titration of hemoglobin A with that of the disubstituted derivative as a function of pH established the contribution of the γ-carboxyl group of Glu-43(β) to the alkaline Bohr effect of the protein. Glu-43(β) is located at the α1\β1 interface of hemoglobin A, and the increase in the pKa of the γ-carboxyl group of Glu-43(β) upon deoxygenation of the protein apparently reflects an increase in the hydrophobicity of the α1\β1 interface of the molecule.",
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T1 - Contribution of the γ-carboxyl group of glu-43(β) to the alkaline bohr effect of Hemoglobin A

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N2 - Glu-43(β) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(β) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the γ-carboxyl group of Glu-43(β) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(β) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pKa of 7.0 for the γ-carboxyl group of Glu-43(β). This pKa is considerably higher than the pKa of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pKa of the γ-carboxyl group of Glu-43(β) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography. The chemical characterization of this derivative of hemoglobin A, involving the separation of the modified β-globin by RPHPLC, isolation of the modified tryptic peptide by RPHPLC, and amino acid sequencing of the modified tryptic peptide, has established that this is a homogeneous derivative of hemoglobin in which both γ-carboxyl groups of Glu-43(β) have been amidated. The amidation of Glu-43(β) increases the O2 affinity slightly without any influence on the Hill coefficient. A comparison of the Bohr proton titration of hemoglobin A with that of the disubstituted derivative as a function of pH established the contribution of the γ-carboxyl group of Glu-43(β) to the alkaline Bohr effect of the protein. Glu-43(β) is located at the α1\β1 interface of hemoglobin A, and the increase in the pKa of the γ-carboxyl group of Glu-43(β) upon deoxygenation of the protein apparently reflects an increase in the hydrophobicity of the α1\β1 interface of the molecule.

AB - Glu-43(β) of hemoglobin A exhibits a high degree of chemical reactivity around neutral pH for amidation with nucleophiles in the presence of carbodiimide. Such a reactivity is unusual for the side-chain carboxyl groups of proteins. In addition, the reactivity of Glu-43(β) is also sensitive to the ligation state of the protein [Rao, M. J., & Acharya, A. S. (1991) J. Protein Chem. 10, 129-138]. The influence of deoxygenation of hemoglobin A on the chemical reactivity of the γ-carboxyl group of Glu-43(β) has now been investigated as a function of pH (from 5.5 to 7.5). The chemical reactivity of Glu-43(β) for amidation increases upon deoxygenation only when the modification reaction is carried out above pH 6.0. The pH-chemical reactivity profile of the amidation of hemoglobin A in the deoxy conformation reflects an apparent pKa of 7.0 for the γ-carboxyl group of Glu-43(β). This pKa is considerably higher than the pKa of 6.35 for the oxy conformation. The deoxy conformational transition mediated increase in the pKa of the γ-carboxyl group of Glu-43(β) implicates this carboxyl group as an alkaline Bohr group. The amidated derivative of hemoglobin A with 2 mol of glycine ethyl ester covalently bound to the protein was isolated by CM-cellulose chromatography. The chemical characterization of this derivative of hemoglobin A, involving the separation of the modified β-globin by RPHPLC, isolation of the modified tryptic peptide by RPHPLC, and amino acid sequencing of the modified tryptic peptide, has established that this is a homogeneous derivative of hemoglobin in which both γ-carboxyl groups of Glu-43(β) have been amidated. The amidation of Glu-43(β) increases the O2 affinity slightly without any influence on the Hill coefficient. A comparison of the Bohr proton titration of hemoglobin A with that of the disubstituted derivative as a function of pH established the contribution of the γ-carboxyl group of Glu-43(β) to the alkaline Bohr effect of the protein. Glu-43(β) is located at the α1\β1 interface of hemoglobin A, and the increase in the pKa of the γ-carboxyl group of Glu-43(β) upon deoxygenation of the protein apparently reflects an increase in the hydrophobicity of the α1\β1 interface of the molecule.

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