Influence of the chemical nature of side chain at β 108 of hemoglobin a on the modulation of the oxygen affinity by chloride ions

Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: Hemoglobins presbyterian and yoshizuka

J. Kevin O'Donnell, Patrick Birch, Cynthia T. Parsons, Steven P. White, Jeannine Okabe, Mike J. Martin, Connie Adams, Karuna Sundarapandiyan, Belur N. Manjula, A. Seetharama Acharya, John S. Logan, Ramesh Kumar

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Hemoglobin A (HbA) and two low oxygen affinity variants of HbA, HbPresbyterian and HbYoshizuka, were produced in transgenic pigs and purified to homogeneity by ion-exchange chromatography. These two variants contain either lysine (HbPresbyterian) or aspartic acid (HbYoshizuka) instead of the normal asparagine residue at position β108 in HbA. Transgenic pigs expressed these variants at a level up to 11% and were healthy. Both HbPresbyterian and HbYoshizuka exhibited low O2 affinity (P50 of 21.2 and 18.9, respectively, compared with control HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooperativity with Hill coefficients of 2.9 and 2.5, respectively. HbPresbyterian exhibited Bohr effect comparable with HbA. In contrast, HbYoshizuka had a diminished response to changes in pH. Thus the structural basis of reduced O2 affinity of these variants appears to be distinct: the consequence of mutation at β108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O2 affinity of the variants to the presence of Cl-. The O2 affinity of HbYoshizuka is insensitive to changes in Cl- concentration, whereas the O2 affinity of HbPresbyterian exhibited a pronounced and dramatic chloride effect. In fact, P50 of HbPresbyterian was identical to that of HbA at very low Cl- concentrations, and the P50 increased to >40 at 0.5 M Cl-. The chloride effect was completely abolished when HbPresbyterian was stabilized at the 2,3-diphosphoglycerate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in HbPresbyterian, Cl- can bridge the ε-amino group of Lysβ108 with either the guanidino group of Argβ104 or the ε-amino group of Lysα99, resulting in the stabilization of the "T" structure. The utility of these low O2 affinity hemoglobins as cell-free oxygen carriers is discussed.

Original languageEnglish (US)
Pages (from-to)27692-27699
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number44
StatePublished - Nov 4 1994

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Hemoglobin A
Chlorides
Hemoglobins
Swine
Modulation
Ions
Oxygen
2,3-Diphosphoglycerate
Molecular modeling
Asparagine
Ion Exchange Chromatography
Chromatography
Aspartic Acid
Lysine
hemoglobin Presbyterian
Ion exchange
Stabilization
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Influence of the chemical nature of side chain at β 108 of hemoglobin a on the modulation of the oxygen affinity by chloride ions : Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: Hemoglobins presbyterian and yoshizuka. / O'Donnell, J. Kevin; Birch, Patrick; Parsons, Cynthia T.; White, Steven P.; Okabe, Jeannine; Martin, Mike J.; Adams, Connie; Sundarapandiyan, Karuna; Manjula, Belur N.; Acharya, A. Seetharama; Logan, John S.; Kumar, Ramesh.

In: Journal of Biological Chemistry, Vol. 269, No. 44, 04.11.1994, p. 27692-27699.

Research output: Contribution to journalArticle

O'Donnell, JK, Birch, P, Parsons, CT, White, SP, Okabe, J, Martin, MJ, Adams, C, Sundarapandiyan, K, Manjula, BN, Acharya, AS, Logan, JS & Kumar, R 1994, 'Influence of the chemical nature of side chain at β 108 of hemoglobin a on the modulation of the oxygen affinity by chloride ions: Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: Hemoglobins presbyterian and yoshizuka', Journal of Biological Chemistry, vol. 269, no. 44, pp. 27692-27699.
O'Donnell, J. Kevin ; Birch, Patrick ; Parsons, Cynthia T. ; White, Steven P. ; Okabe, Jeannine ; Martin, Mike J. ; Adams, Connie ; Sundarapandiyan, Karuna ; Manjula, Belur N. ; Acharya, A. Seetharama ; Logan, John S. ; Kumar, Ramesh. / Influence of the chemical nature of side chain at β 108 of hemoglobin a on the modulation of the oxygen affinity by chloride ions : Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: Hemoglobins presbyterian and yoshizuka. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 44. pp. 27692-27699.
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abstract = "Hemoglobin A (HbA) and two low oxygen affinity variants of HbA, HbPresbyterian and HbYoshizuka, were produced in transgenic pigs and purified to homogeneity by ion-exchange chromatography. These two variants contain either lysine (HbPresbyterian) or aspartic acid (HbYoshizuka) instead of the normal asparagine residue at position β108 in HbA. Transgenic pigs expressed these variants at a level up to 11{\%} and were healthy. Both HbPresbyterian and HbYoshizuka exhibited low O2 affinity (P50 of 21.2 and 18.9, respectively, compared with control HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooperativity with Hill coefficients of 2.9 and 2.5, respectively. HbPresbyterian exhibited Bohr effect comparable with HbA. In contrast, HbYoshizuka had a diminished response to changes in pH. Thus the structural basis of reduced O2 affinity of these variants appears to be distinct: the consequence of mutation at β108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O2 affinity of the variants to the presence of Cl-. The O2 affinity of HbYoshizuka is insensitive to changes in Cl- concentration, whereas the O2 affinity of HbPresbyterian exhibited a pronounced and dramatic chloride effect. In fact, P50 of HbPresbyterian was identical to that of HbA at very low Cl- concentrations, and the P50 increased to >40 at 0.5 M Cl-. The chloride effect was completely abolished when HbPresbyterian was stabilized at the 2,3-diphosphoglycerate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in HbPresbyterian, Cl- can bridge the ε-amino group of Lysβ108 with either the guanidino group of Argβ104 or the ε-amino group of Lysα99, resulting in the stabilization of the {"}T{"} structure. The utility of these low O2 affinity hemoglobins as cell-free oxygen carriers is discussed.",
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T1 - Influence of the chemical nature of side chain at β 108 of hemoglobin a on the modulation of the oxygen affinity by chloride ions

T2 - Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: Hemoglobins presbyterian and yoshizuka

AU - O'Donnell, J. Kevin

AU - Birch, Patrick

AU - Parsons, Cynthia T.

AU - White, Steven P.

AU - Okabe, Jeannine

AU - Martin, Mike J.

AU - Adams, Connie

AU - Sundarapandiyan, Karuna

AU - Manjula, Belur N.

AU - Acharya, A. Seetharama

AU - Logan, John S.

AU - Kumar, Ramesh

PY - 1994/11/4

Y1 - 1994/11/4

N2 - Hemoglobin A (HbA) and two low oxygen affinity variants of HbA, HbPresbyterian and HbYoshizuka, were produced in transgenic pigs and purified to homogeneity by ion-exchange chromatography. These two variants contain either lysine (HbPresbyterian) or aspartic acid (HbYoshizuka) instead of the normal asparagine residue at position β108 in HbA. Transgenic pigs expressed these variants at a level up to 11% and were healthy. Both HbPresbyterian and HbYoshizuka exhibited low O2 affinity (P50 of 21.2 and 18.9, respectively, compared with control HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooperativity with Hill coefficients of 2.9 and 2.5, respectively. HbPresbyterian exhibited Bohr effect comparable with HbA. In contrast, HbYoshizuka had a diminished response to changes in pH. Thus the structural basis of reduced O2 affinity of these variants appears to be distinct: the consequence of mutation at β108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O2 affinity of the variants to the presence of Cl-. The O2 affinity of HbYoshizuka is insensitive to changes in Cl- concentration, whereas the O2 affinity of HbPresbyterian exhibited a pronounced and dramatic chloride effect. In fact, P50 of HbPresbyterian was identical to that of HbA at very low Cl- concentrations, and the P50 increased to >40 at 0.5 M Cl-. The chloride effect was completely abolished when HbPresbyterian was stabilized at the 2,3-diphosphoglycerate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in HbPresbyterian, Cl- can bridge the ε-amino group of Lysβ108 with either the guanidino group of Argβ104 or the ε-amino group of Lysα99, resulting in the stabilization of the "T" structure. The utility of these low O2 affinity hemoglobins as cell-free oxygen carriers is discussed.

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