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Kinetic and equilibrium studies on low and high affinity hemoglobins
Abstract
Functional characteristics of the allosteric R- and T-states of hemoglobin, modeled by diverse hemoglobins, are examined using ligand binding kinetic and equilibrium studies. The kinetics and thermodynamics of oxygen and CO ligation have been determined for the tetrameric hemoglobin from Urechis caupo, a model T-state Hb. $\Delta$S$\sp\circ$ for O$\sb2$ and CO ligation corresponds to simple immobilization of the ligand; the low ligand affinities thus derive from small $\Delta$H$\sp\circ$s, which are correlated with the large (0.29 A) out of plane displacement of the Fe shown by EXAFS studies. A direct measurement of oxygen binding gave a Hill number of 1.3. The half-time for the T* $\to$ T transition is 8-14 ms at 20$\sp\circ$C. The kinetics and thermodynamics of oxygen and CO binding have been determined for the eight components of soybean leghemoglobin, a model for R-state Hb. $\Delta$S$\sp\circ$ for O$\sb2$ and CO ligation are virtually the same as those determined for Urechis Hb. The large $\Delta$H$\sp\circ$s of ligation are consistent with EXAFS studies showing the Fe to be in the mean plane of the heme in the Lb-CO complex, and account for the high affinity of Lb. Significant kinetic differences among the components must derive from amino acid substitutions distant from the heme pocket. Kinetic data from both the low and high affinity conformers of carp Hb are fit extensively to an allosteric model. The unliganded R-state is best modeled by three states, and a minimum of five unliganded states are required to accommodate the carp data overall. A precise value for the tetramer-dimer equilibrium consent of human Hb was obtained, using a multipass cuvette constructed to allow measurements on dilute (0.25 $\mu$M) Hb solutions. This K$\sb{\rm TD}$ value was utilized in a study of the kinetics and thermodynamics of binding the last molecule of oxygen to tetramers and dimers of valency hybrids of human Hb. Tetramers of both alpha and beta hybrids had an apparent enhanced affinity over dimers for this oxygen, of 280-360 cal/mole.
Subject Area
Biochemistry
Recommended Citation
Parkhurst, Kay Martin, "Kinetic and equilibrium studies on low and high affinity hemoglobins" (1991). ETD collection for University of Nebraska-Lincoln. AAI9121933.
https://digitalcommons.unl.edu/dissertations/AAI9121933