Hemoglobin tetramers stabilized by a single intramolecular cross-link

Abstract

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.