Chemical Modification of Hemoglobin with 4,4′-Diisothiocyanostilbene-2,2′-Disulfonate (DIDS): Initial Studies of a Potential Blood Substitute

Abstract

Several bifunctional 2,3-diphosphoglycerate (DPG) analogs will cross-link residues in the DPG binding site and prevent subunit dissociation of undenatured hemoglobin. By selecting reagents of proper structure and appropriate reaction conditions, it is possible to produce modified hemoglobins with oxygen equilibria shifted towards either the low affinity or the nigh affinity state. One such reagent is 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). Depending upon the specific reaction conditions, different DIDS products of hemoglobin result. The major deoxyhemoglobin reaction product is a molecule in which one DIDS per tetramer is incorporated, cross-linking between the two β chain amino terminal valyl residues. This derivative, Hb(DIDS)-T, has greatly decreased O₂ affinity with a P₅₀ of 24 mmHg compared to P₅₀ of 5 mmHg for normal hemoglobin (50 mM bis-Tris, pH 7.4, 0.1 M Cl-, and 25°C). Its cooperativity is reduced moderately (nmax = 2.1) but the addition of 2,3-DPG has no effect on its oxygen affinity. In contrast, the predominant product obtained by reacting oxy-or carbonmonoxyhemoglobin with DIDS is a uncross-linked hemoglobin containing two molecules of DIDS per tetramer resulting from separate adduct formation with each β chain amino terminal valyl residues. The O₂ affinity of this derivative, Hb(DIDS)₂-R, is increased greatiy to a P₅₀ of 1.4 mmHg. Other cross-linked and uncross-linked produr. s of DIDS reacted deoxyhemoglobin are also formed including some Hb(DIDS)₂-R. Preliminary studies of vascular retention of a mixture of DIDS-hemoglobin products in the rat indicate that at least three derivatives are still retained after 4 hours of circulation by which time unmodified and several presumably uncross-linked derivatives have been cleared.