Kinetic Studies on the Five Principal Components of Normal Adult Human Hemoglobin

Robert C. Steinmeier, Lawrence J Parkhurst

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

The five principal components of human hemoglobin (A1a, A1b, A1c, A0, and A2) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A0and A2are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A0is greatly reduced in A1aand A1cand is essentially absent in A1b- The general effect of p-mercuribenzoate binding on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin A1ais anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1l′) showed the greatest variation among the five hemoglobins, with A0having the smallest constant, and A1bthe largest. For the native hemoglobins, 1l′ for A1bwas more than twice that for A0; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH from 7 to 8 increases 1l′ for all hemoglobins, but A1ais anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A0, the fastest, and A1a, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for A1aand is 4.4 times that for A0, and 5.6 times that for A1c, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for A1aand A1b, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN-and N3 -binding to the five methemoglobins.

Original languageEnglish (US)
Pages (from-to)1564-1572
Number of pages9
JournalBiochemistry
Volume14
Issue number8
DOIs
StatePublished - Apr 1 1975

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Hemoglobins
Carbon Monoxide
Kinetics
Association reactions
Oxygen
Methemoglobin
Oxyhemoglobins
Column chromatography
Molecules
Photolysis
Equilibrium constants
Isoelectric Point
Isoelectric Focusing
Kinetic parameters
varespladib methyl
Dimers
Conformations
Chromatography
Rate constants
Gels

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic Studies on the Five Principal Components of Normal Adult Human Hemoglobin. / Steinmeier, Robert C.; Parkhurst, Lawrence J.

In: Biochemistry, Vol. 14, No. 8, 01.04.1975, p. 1564-1572.

Research output: Contribution to journalArticle

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N2 - The five principal components of human hemoglobin (A1a, A1b, A1c, A0, and A2) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A0and A2are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A0is greatly reduced in A1aand A1cand is essentially absent in A1b- The general effect of p-mercuribenzoate binding on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin A1ais anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1l′) showed the greatest variation among the five hemoglobins, with A0having the smallest constant, and A1bthe largest. For the native hemoglobins, 1l′ for A1bwas more than twice that for A0; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH from 7 to 8 increases 1l′ for all hemoglobins, but A1ais anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A0, the fastest, and A1a, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for A1aand is 4.4 times that for A0, and 5.6 times that for A1c, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for A1aand A1b, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN-and N3 -binding to the five methemoglobins.

AB - The five principal components of human hemoglobin (A1a, A1b, A1c, A0, and A2) have been isolated by column chromatography and by preparative isoelectric focusing in gels. The isoelectric points and a number of kinetic parameters have been determined for each hemoglobin. The greatest kinetic differences are found in the binding of CO to the deoxy conformation. At pH 7, A0and A2are nearly identical in their overall reaction with CO, whereas the initial lag phase characteristic of crude hemolysate and A0is greatly reduced in A1aand A1cand is essentially absent in A1b- The general effect of p-mercuribenzoate binding on CO association is to magnify kinetic differences among the hemoglobins, diminish the initial lag phase, and increase the overall rate of CO binding. Hemoglobin A1ais anomalous in that the overall CO binding rate actually decreases after reaction with the mercurial. In terms of an Adair model with four association constants the rate constant for the binding of the first molecule of CO (1l′) showed the greatest variation among the five hemoglobins, with A0having the smallest constant, and A1bthe largest. For the native hemoglobins, 1l′ for A1bwas more than twice that for A0; for the mercurated hemoglobins, the difference was greater than threefold. Raising the pH from 7 to 8 increases 1l′ for all hemoglobins, but A1ais anomalous in having a slower overall rate for CO binding at the higher pH. At pH 9, the time course of CO binding is biphasic for all hemoglobins, with A0, the fastest, and A1a, the slowest, differing by nearly threefold in rate. The equilibrium constant for the tetramer-dimer equilibrium was determined by flash photolysis. The largest dissociation constant occurs for A1aand is 4.4 times that for A0, and 5.6 times that for A1c, the least dissociated of the hemoglobins. The overall oxygen dissociation reaction is biphasic for A1aand A1b, with the two phases differing by a factor of 5; the dissociation reactions for the other three hemoglobins appear essentially monophasic. The kinetics of dissociation of the first oxygen molecule from oxyhemoglobin are very similar for all five hemoglobins, as are the association kinetics for CN-and N3 -binding to the five methemoglobins.

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