Subunit dissociations in natural and recombinant hemoglobins

Abstract

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

“…As controls, we examined the gel filtration elution profiles of the reduced CO forms of HbA and rHb Kirklareli as a function of total protein concentration using the methods described by Manning et al (19). The K 4,2 for HbCO Kirklareli was estimated to be ϳ0.3 M, which is ϳ4-fold smaller than the value estimated for HbA in our experiments, 1.1 M. The latter value is very similar to previously reported K 4,2 values (20,21). Thus, the rapid rates of degradation of Hb Kirklareli are not due to increased dissociation into dimers.…”

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces

“…In fact, the prevailing view is that all normal human Hbs are structurally alike and there is little consideration of strong or weak subunit interactions as being of importance. 7,8 However, using direct and very sensitive techniques, 9,10 we found large differences among them regarding the structural strengths at their subunit interfaces as shown below by their strikingly different patterns during molecular sieve chromatography (gel filtration) when performed at concentrations that do not obscure these intrinsic differences, that is, much lower than their equilibrium constants. These gel filtration patterns represent distinct phenotypes for the embryonic, fetal, and adult Hbs that appear to be linked to the developmental process.…”

“…9,10 The applied Hb concentrations were in the range of 10-100 nM, which is near or below the tetramer-dimer dissociation constants for all the Hbs shown in Figure 2. Hence, the tetrameric state would not predominate thus favoring the possibility of detecting dimer dissociation to monomers especially for weak dimers.…”

“…9,10 The embryonic human Hbs were purified from the blood of transgenic mice 12,13 that had been transfected with the human embryonic globin genes. 23 All Hbs were homogenous as determined by high-resolution isoelectric focusing and analytical FPLC; each had the correct mass as measured by mass spectrometry.…”

Determination of the Tetramer-Dimer Equilibrium Constant of Rabbit Hemoglobin

“…This is so because, it is known that Cys F9 [93] β of hemoglobin dimers react faster than in tetramers 13 . Therefore, the trend of k seen in figures 2 to 5 arises because as the hemoglobin concentration increases, the proportion of dimers decreases.…”