Development of antisickling compounds that chemically modify hemoglobin S specifically within the 2,3-diphosphoglycerate binding site

Abstract

In this paper we describe a class of affinity reagents that react specifically with hemoglobin S at the 2,3-diphosphoglycerate binding site and effectively disrupt the sickling process. The prototype of these compounds is the bifunctional acylating agent bis(3,5-dibromosalicyl) fumarate. Previously it was shown that this compound reacts selectively with oxyhemoglobin to cross-link the β chains of the tetramer ( Walder et al., 1979 a ). The site of cross-linking has now been established by X-ray crystallographic studies to be from Lys82 β 1 to Lys82 β 2, spanning the DPG ‡ ‡ Abbreviations used: DPG, 2,3-diphosphoglycerate; p 50, partial pressure of oxygen needed to half-saturate hemoglobin; Hb, hemoglobin. binding site. The oxygen binding properties of the cross-linked hemoglobin are very similar to those of native hemoglobin A in the absence of DPG. However, in the presence of DPG, the oxygen affinity of the cross-linked hemoglobin is increased due to the blockade of the DPG site by the interchain bridge. This effect may indirectly interfere with the sickling process in vivo. More importantly the cross-link modification directly inhibits the polymerization of deoxyhemoglobin S. The solubility of the cross-linked derivative is increased by approximately 35% relative to that of native deoxyhemoglobin S. The X-ray crystallographic studies reveal that the cross-link strongly perturbs the acceptor site for Val6β in the lateral contact between molecules of deoxyhemoglobin S within the double strand of the sickle cell fiber. Our current hypothesis is that this structural perturbation weakens the lateral contact and is responsible for the observed increase in solubility. Due to the hydrophobic properties of the halogen substituents bis(3,5-dibromosalicyl) fumarate is able to traverse the red-cell membrane and, therefore, may be active in vivo. These studies provide the basis for further design and development of affinity reagents directed to the DPG binding site that may prove useful in the clinical management of sickle cell disease.