Cross-linking reagents for the specific modification of human hemoglobin have been developed and reported previously. The cross-linked protein can potentially be used as a red cell substitute and also as a carrier in bioconjugation. Cross-linkers with structurally defined bridging moieties and highly selective reaction sites can produce specifically defined linkages in the protein. This permits altered properties of the modified protein to be clearly related to its structure.
Hemoglobin in the red blood cell consists essentially of four sub-units, two .alpha.-units and two .beta.-units, each such unit including a heme molecule. Outside the red cell, this tetrameric hemoglobin tends to dissociate into .alpha..beta. dimeric sub-units, and even further into monomeric sub-units. Such sub-units resulting from dissociation, of molecular weight c. 16,000 or 32,000, are too small for safe and effective use as a blood substitute in the mammalian system, since they tend to be excreted by the kidneys during circulation, and contribute to renal failure. Accordingly, it is usual to use hemoglobin, in a blood substitute, with chemical crosslinks formed between the selected subunits to bind the hemoglobin in its complete form, of molecular weight c.64,000, i.e. intramolecularly crosslinked hemoglobin. A variety of chemical crosslinking reagents have been proposed for this purpose.
A few of the crosslinking reagents previously proposed for intramolecular crosslinking of hemoglobin also cause incidental intermolecular crosslinking thereof, to form bis tetrameric hemoglobin of molecular weight c.128,000, tetrakis tetrameric hemoglobin of molecular weight c.256,000, and so on, up to species with molecular weight in excess of 1,000,000. These processes produce a mixture of such species, the reaction product thus having a wide molecular weight distribution. The optimum molecular weight, or molecular weight distribution, for hemoglobin for use as a blood substitute in mammals is still subject to study. Control over the molecular weight and molecular weight distribution is important, and this in turn means that control over processes of crosslinking hemoglobin, to yield a single species of crosslinked hemoglobin or a mixture containing known and controllable quantities of different species, is important.