Blood substitutes comprising hemoglobin and methods of making

A method of preparing a blood substitute preparation comprising hemoglobin, the method comprising:

• (i) isolating erythrocytes from blood;
• (ii) isolating a low purity erythrocyte protein fraction comprising hemoglobin protein molecules and endogenous non-hemoglobin protein complement from the erythrocytes, the low purity erythrocyte protein fraction comprising from at least about 0.2% (mole/mole) up to about 20% (mole/mole) of the endogenous non-hemoglobin protein complement, the endogenous non-hemoglobin protein complement comprising carbonic anhydrase;
• and
• (iii) contacting the low purity erythrocyte protein fraction with a reactant capable of chemically modifying the proteins in the protein fraction, the reactant thereby mediating the formation of cross-linked proteins comprising intermolecular cross-linkages between the hemoglobin protein molecules, and intermolecular cross-linkages between the hemoglobin protein molecules and the endogenous non-hemoglobin protein complement, to thereby form a blood substitute preparation.