Functional Comparison of Specifically Cross-Linked Hemoglobins Biased Toward the R and T States

Abstract

Selected functional and spectroscopic properties of two human hemoglobin (HbA 0) derivatives that were site-specifically cross-linked in the cleft between β-chains where 2,3-bisphosphoglycerate normally binds have been determined to assess the effects of the cross-linking on the behavior of the protein. Trimesoyl tris(3,5-dibromosalicylate) (TTDS) cross-links Hb between β82Lys residues. The resulting TTDS-Hb exhibits a slower rate of oxygen dissociation and an increased rate of carbon monoxide association than observed for HbA 0. The electron paramagnetic resonance (EPR) spectrum of TTDS-HbNO does not exhibit the hyperfine structure that is indicative of significant conformational change despite the fact that the 2,3-bisphosphoglycerate binding site is occupied by the cross-linking reagent. The reactivity of the β93Cys residues of TTDS-Hb is only slightly decreased relative to that of HbA 0. On the other hand, cross-linking Hb between Lys82 and the amino-terminal β1Val group with trimesoyl tris(methyl phosphate) (TMMP) increases the rate of oxygen dissociation and reduces the rate of CO association relative to the rates observed for HbA 0. In addition, the EPR spectrum of the TMMP-HbNO exhibits the three-line hyperfine structure that results from disruption of the proximal His-Fe bond of the α-chains, and the accessibility of the βCys93 residues in this derivative is decreased fourfold. The present results are consistent with the conclusion that the quaternary structure of TTDS-Hb is shifted toward the R state whereas the quaternary structure of TMMP-Hb is shifted toward the T state and provides additional evidence that the identity of the residues involved in intramolecular cross-linking of hemoglobin within the 2,3-bisphosphoglycerate binding site between β-chains can have a significant influence on the conformational and functional properties of the protein.