Affinity Labeling of Hemoglobin with 4,4′-Diisothiocyanostilbene-2,2^/-disulfonate: Covalent Cross-Linking in the 2,3-Diphosphoglycerate Binding Site

The bifunctional reagent 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) reacts with hemoglobin to give various products whose properties are dependent on the ligation state of the protein during the reaction. A major product obtained after reaction of (carbonmonoxy)hemoglobin with DIDS was a high oxygen affinity derivative [P₅₀= 1.4 mmHg, control P₅₀= 6 mmHg; 50 mM [bis(2-hydroxyethyl)-amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.4, 0.1 M Cl^-, 25 °C] which contained two molecules of DIDS per tetramer resulting from adduct formation at each β-chain amino terminus. In contrast, a major product of the reaction of deoxyhemoglobin with DIDS consisted of hemoglobin which had incorporated one molecule of DIDS per tetramer and was cross-linked between the β-chain amino termini. This cross-linked hemoglobin was found to have a greatly decreased 0₂affinity (P₅₀= 28 mmHg). Inhibition of the T to R transition due to the structural constraint produced by cross-linking the β amino termini is likely to be a major factor in the decreased 0₂affinity of this product. The structural and functional properties of this molecule make it a potential candidate for a cell-free blood substitute.