Kinetic and Equilibrium Studies on the Role of the β-147 Histidine in the Root Effect and Cooperativity in Carp Hemoglobin

Lawrence J Parkhurst, Dixie J. Goss, Max F. Perutz

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Abstract

In fish possessing a swim bladder, the alkaline Bohr effect is much enhanced. At pH ≤6, oxygen binding becomes noncooperative and the oxygen affinity very low. This phenomenon is known as the Root effect. In mammalian hemoglobin a major fraction of the alkaline Bohr effect is contributed by His-HC3(146)β. Perutz proposed that the Root effect is a consequence mainly of the replacement of Cys-F9(93)β in mammalian hemoglobin by Ser in fish hemoglobin. Model building showed that this Ser would stabilize the salt bridges between His-HC3 and Asp- or Glu-FG1 in the T structure by the formation of two additional hydrogen bonds. If this theory is correct, then enzymatic cleavage of His-HC3β should inhibit the Root effect. We have prepared des-His-HC3(147)β carp hemoglobin and compared its ligand-binding equilibria and kinetics with those of native carp hemoglobin and have interpreted the results in terms of the allosteric model. Our results show that removal of His-HC3(147)β halves the alkaline Bohr effect as determined from ∂ log P50/∂pH, accelerates the binding of CO at acid pH, and diminishes the pH dependence of the CO on rate. In native carp hemoglobin at pH 7, recombination of CO after photolytic dissociation of only part of the CO is faster than after full dissociation. In des-His carp hemoglobin at pH 7, this acceleration is not observed. These results show that removal of His-HC3 inhibits all the phenomena associated with the Root effect. Our measurements indicate that this is due to a destabilization of the T structure by the equivalent of 3.1 kcal/mol of tetramer.

Original languageEnglish (US)
Pages (from-to)5401-5409
Number of pages9
JournalBiochemistry
Volume22
Issue number23
DOIs
Publication statusPublished - Jan 1 1983

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ASJC Scopus subject areas

  • Biochemistry

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