Abstract
Five different human hemoglobins were used to test the postulate that dissociation of hemoglobin (Hb) tetramers into alpha beta dimers and dissociation of heme from globin are linked reactions. Spectrophotometric measurements of the initial rate of heme transfer from Hb to serum albumin were made over a 3000-fold range of Hb concentration and yielded the heme-globin dissociation rate constant for tetramers and that for dimers. The tetramer-dimer dissociation constant (K4,2) could then be calculated from the rate constant at intermediate concentrations. The values obtained for the five hemoglobins, spanning a 250-fold range in K4,2, were in good agreement with those found by direct methods. The relation between this new linkage reaction of hemoglobin and the classical ones, such as the reciprocal relation between the binding of oxygen and protons, is discussed briefly.
Highlights
From the Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, New York 10032
Spectrophotometric measurements of the initial rate of heme transfer from Hb to serum albumin were made over a 3000-fold range of Hb concentration and yielded the heme-globin dissociation rate constant for tetramers and that for dimers
In the course of developing a spectrophotometric method for measuring the rate and equilibrium of heme exchange between Hb and serum albumin, we found recently [3] that heme transfer from Hb dimers is very much faster than that from tetramers
Summary
(Received for publication, February 9, 1995, and in revised form, March 27, 1995). From the Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, New York 10032. The values obtained for the five hemoglobins, spanning a 250-fold range in K4 2, were in good agreement with those found by direct ~ethods The relation between this new linkage reaction of hemoglobin and the classical ones, such as the reciprocal relation between the binding ofoxygen and protons, is discussed briefly. In the course of developing a spectrophotometric method for measuring the rate and equilibrium of heme exchange between Hb and serum albumin, we found recently [3] that heme transfer from Hb dimers is very much faster than that from tetramers This suggested that binding of heme to globin and binding of af dimers to each other are yet another example of linked functions. In this report we present evidence that this is the case and that measurements of the rate of heme transfer as a function of the Hb concentration can be used to determine the tetramer-dimer dissociation constant of normal, mutant, and chemically modified hemoglobins
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