EFFECTS OF INTRAMOLECULAR AND INTERMOLECULAR CROSS-LINKING ON THE FREE-RADICAL REACTIONS OF BOVINE HEMOGLOBINS

Chemical modifications of human or bovine hemoglobins are designed to produce proteins that can act as oxygen-carrying blood substitutes. Co ncerns about the redox reactivity of cell-free hemoglobin and its cont ribution to tissue-damaging oxygen free radicals has not been fully es tablished. We determined that bovine hemoglobins intra- or intermolecu larly crosslinked differ in their ability to generate or interact with reactive oxygen species. These differences do not correlate with thei r oxygen affinities. We compared HbBv-FMDa, produced by the reaction o f bovine hemoglobin with fumaryl-monodiaspirin and Poly HbBv, a glutar aldehyde polymerized bovine hemoglobin, with unmodified bovine hemoglo bin (HbBv). Superoxide radicals are produced during the spontaneous ox idation of hemoglobin. Relative to the other two proteins, Poly HbBv w as found to be more susceptible to autoxidation. Spectral changes indi cative of protein modification and ferrylhemoglobin formation during t he enzymatic peroxidation of these hemoglobins differ qualitatively an d occur at an increasing order, poly HbBv > HbBv > HbBv-FMDA. The prot eins also differ in the rate of hemoglobin catalyzed NADPH oxidation a nd aniline hydroxylation, reactions mediated by reactive oxygen specie s. Taken together, our results and those reported previously on modifi ed human hemoglobins, suggest that redox and oxygen-carrying functions of hemoglobin can be experimentally manipulated as independently sele ctable parameters that may ultimately aid in the design of a safer rep erfusion agent.