Human Hemoglobin Anaerobically Reacted with Divinyl Sulfone: A Source for Oxygen Carriers

Abstract

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°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°C and pH 7.4. From gelpermeation 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 ∼15 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 transrusional fluids.