Properties of a Novel Superoxide Dismutase-Hemoglobin Conjugate of Possible Value in Blood Substitute Formulation

Abstract

Because of concerns about blood-borne diseases, there is a continuing interest in the development of a temporary blood replacement for alleviating the harmful effects of extensive blood loss. It has long been recognized that to develop a carrier capable of delivering oxygen to tissues based on hemoglobin, the chemical modification of hemoglobin is necessary. Unmodified hemoglobin has too short a retention time in circulation and too high an oxygen affinity to be useful in treating hemorrhagic shock. Therefore cross-linking of hemoglobin subunits or molecules has received considerable attention as a basis for the formulation of a safe efficacious blood substitute. Both native stroma free hemoglobin and chemically modified forms of hemoglobin have been shown to facilitate the formation of potentially harmful forms of oxygen, although the extent of this varies with the form of the protein (Alayash, 1995). The conditions under which administration of a hemoglobin-based oxygen carrier would be advised are those which appear to be associated with tissue injury accompanying reperfusion. It is thought that tissue damage occurs following treatment of hemorrhagic shock because of a burst of formation of toxic forms of oxygen accompanying oxygen resupply to tissues (Granger and Korthius, 1995; Biro et al., 1995). The inclusion of features which would be expected to alleviate or prevent the generation of harmful forms of oxygen would therefore seem valuable in developing therapeutic formulations. The following work describes a novel chemical modification which can be used to make a modified form of hemoglobin containing an enzyme which functions physiologically to prevent the accumulation of harmful forms of oxygen. Superoxide dismutase when eovalently joined to hemoglobin has been shown to have an extended lifetime in circulation. The modified enzyme when injected into rats has a half-life in circulation in an active form which might be expected to be of benefit with respect to minimizing reperfusion injury following resuscitation with a hemoglobin-based blood substitute.