Suzanna Kwong

Oxygen affinity as an index of hemoglobin S polymerization: A new micromethod☆

Abstract The decrease in oxygen affinity with increasing hemoglobin concentration, which occurs in solutions of pure hemoglobin S, can be used to determine the minimum concentration at which polymerization of the deoxy form takes place. On this basis a very sensitive method for measuring the minimum gelling concentration has been developed. The influence of temperature, pH, and other hemoglobins on the end points obtained by this method is described. In all cases excellent agreement with the minimum gelling concentration determined directly on larger samples was observed. The results of this investigation demonstrate that the decrease in oxygen affinity of red cells containing hemoglobin S with increasing intracorpuseular hemoglobin concentration [May, A., and Huehns, E. R. (1975) Brit. J. Haematol.30, 317] is a direct consequence of the gelling properties of hemoglobin S alone.

Hemoglobin tetramers stabilized by a single intramolecular cross-link

A specific intramolecular cross-link was introduced into bovine and human hemoglobin by reaction of the deoxyhemoglobin with the dialdehyde, bispyridoxal tetraphosphate (bisPL)P4, followed by reduction with NaBH4. The yield of cross-linked hemoglobin is 80% in both cases, using 1 mol of (bisPL)P4 per mol of Hb. The crosslink is confined to the β chains, where it connects the N-terminal residue (valine and methionine, respectively) to a lysine on the other β chain across the central cavity. The stereochemical requirements for the reaction were probed by using a rigid analogous cross-linking reagent, as well as with a mutant Hb, which has a shorter distance between the residues to which the cross-link is attached. Introduction of the cross-link into human and bovine Hb results in a five-fold and four-fold reduction in the oxygen affinity and a decrease in the Bohr Effect by 1/3 and 1/2, respectively. Oxygenation remains cooperative, albeit with a decreased Hill coefficient. The cross-linked hemoglobins are oxidized more rapidly to the ferric form, but their resistance to heat denaturation is increased. The stability of the link between the β chains and their hemes is 10 times greater in both cross-linked hemoglobins that in their native counterparts. The possible application of this chemical modification for the preparation of hemoglobin-based blood substitutes is discussed.

Intermolecular effects in the polymerization of hemoglobin S.

Myoglobin and isolated α and β subunits of hemoglobin A were found to have the same effect on the polymerization of hemoglobin S as intact hemoglobin A. This demonstrates that copolymerization of hemoglobins A and S need not be invoked to explain the gelling behavior of such mixtures, but that it can be adequately accounted for by the effect of the added hemoglobin on the activity coefficient of hemoglobin S. In addition, a hemoglobin tetramer in which an αAβA dimer is covalently linked to an αAβS dimer between the β chains was found to gel on deoxygenation. Therefore, only one β6 valine per tetramer is necessary for polymerization.

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.

Changes in the functional properties of bovine hemoglobin induced by covalent modification with polyethylene glycol.

Polyethylene glycol conjugation to proteins and peptides (PEGylation) has been shown to promote increased retention time in the circulation as well as to blunt immune or allergic reactions. PEGylated bovine hemoglobin (PEG-Hb) is being explored in human clinical trials as an oxygen delivering agent for the sensitization of solid tumors to radiation therapy. In this study the functional properties of PEG-Hb were compared to those of bovine hemoglobin (Hb), the mutant human hemoglobin Rothchild and bovine hemoglobin crosslinked between the beta chains. The rate of heme transfer from Hb to serum albumin at pH 9.0 was greatly increased by PEGylation, suggesting destabilization of the heme-globin linkage and of the bonds between alpha beta dimers. Measurement of oxygen binding equilibrium showed that the oxygen affinity of Hb became unusually dependent on temperature and Hb concentration after PEGylation. Evidence is presented to suggest that PEGylation of lysine beta-81 at the entrance to the central cavity of the Hb tetramer might be responsible for these observations. The alterations of the functional properties of Hb induced by PEGylation are consistent with the beneficial effects of PEG-Hb in exchange transfusion and radiation sensitization models of human conditions.