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 degrees C] which contained two molecules of DIDS per tetramer resulting from adduct formation at each beta-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 beta-chain amino termini. This cross-linked hemoglobin was found to have a greatly decreased O2 affinity (P50 = 28 mmHg). Inhibition of the T to R transition due to the structural constraint produced by cross-linking the beta amino termini is likely to be a major factor in the decreased O2 affinity of this product. The structural and functional properties of this molecule make it a potential candidate for a cell-free blood substitute.