Human hemoglobin anaerobically reacted with divinyl sulfone: a source for oxygen carriers

Human hemoglobin (HbA) was reacted in anaerobic conditions with divinyl sulfone (DVS). The structural and oxygenation properties of the resulting chemically-modified product were studied in order to assess its potential as a physiological oxygen carrier. The reaction was carried out anaerobically at 25 degrees C and pH 7.4 for 3 h. Quenching was performed with lysine-HCl solution and the resulting solution dialysed to remove unbound DVS and excess lysine. The product, designated Poly HbA-DVS, was characterized structurally using gel-permeation HPLC and SDS-PAGE and functionally employing a Hemox-analyzer at 37 degrees C and pH 7.4. From gel-permeation HPLC it was estimated that about 60% of the starting material was polymerized, with a molecular mass range from 130 to about 500 kDa, and about 40% remained monomeric with a molecular mass of 64 kDa. The virtual absence of a 32 kDa band from the SDS-PAGE pattern of the last eluting HPLC peak and the oxygenation properties of this peak material (P50 = 33 mm Hg, n = 2.2; P50 very different from the approximately 1.5 mm Hg associated with native HbA solution) indicated that the monomeric (64 kDa) component was modified, but virtually noncrosslinked, within the tetramer. The product solution, Poly HbA-DVS, had a P50 of 35 mm Hg, a Hill coefficient n of 1.8 and a methemoglobin content of 5-7%. This material has characteristics appropriate for an oxygen carrier, and can probably be used as such in perfusional and transfusional fluids.