HUMAN HEMOGLOBIN ANAEROBICALLY REACTED WITH DIVINYL SULFONE - A SOURCE FOR OXYGEN CARRIERS

Human hemoglobin (HbA) was reacted in anaerobic conditions with diviny l sulfone (DVS). The structural and oxygenation properties of the resu lting 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 pe rformed with lysine-HCl solution and the resulting solution dialysed t o remove unbound DVS and excess lysine. The product, designated Poly H bA-DVS, was characterized structurally using gel-permeation HPLC and S DS-PAGE and functionally employing a Hemox-analyzer at 37 degrees C an d pH 7.4. From gel-permeation HPLC it was estimated that about 60% of the starting material was polymerized, with a molecular mass range fro m 130 to about 500 kDa, and about 40% remained monomeric with a molecu lar 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 propert ies of this peak material (P-50 = 33 mm Hg, n = 2.2; P-50 very differe nt from the similar to 15 mm Hg associated with native HbA solution) i ndicated that the monomeric (64 kDa) component was modified, but virtu ally noncrosslinked, within the tetramer. The product solution, Poly H bA-DVS, had a P-50 of 35 mm Hg, a Hill coefficient n of 1.8 and a meth emoglobin content of 5-7%. This material has characteristics appropria te for an oxygen carrier, and can probably be used as such in perfusio nal and transfusional fluids.