Conjugating Hemoglobin and Albumin by Strain-Promoted Azide- Alkyne Cycloaddition

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each beta-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier. Strain-promoted azide-alkyne cycloaddition (SPAAC) of site-selectively cross-linked human hemoglobin with specifically modified human serum albumin yields a structurally defined protein-protein conjugate. A circulating solution of the conjugate can provide cooperative oxygen transport and maintain colloid osmotic pressure. image