A core–shell ensemble of bovine hemoglobin (Hb) and human serum albumin (HSA) is an artificial O2 carrier as a red blood cell substitute. This protein particle is created by covalent wrapping of a carbonyl Hb with HSAs: HbR–HSA 3 cluster, where HbR signifies the use of carbonyl Hb (relaxed (R) state conformation) as a starting material. The HbR–HSA 3 cluster exhibits high O2 affinity and low cooperativity. Analysis of the quaternary structure of the central HbR in the cluster revealed that its high O2 affinity is attributed to the physically immobile HbR nucleus. Circular dichroism and UV–vis absorption spectroscopy showed that the structure of deoxy HbR core closely resembles the R-state. The crystal structure of Lys-modified carbonyl HbR was superimposed on that of carbonyl Hb. These results imply that chemical modifications of the surface Lys groups and Cys-93­(β) of the carbonyl Hb with cross-linking agent interfered in the quaternary structure movement from the R-state to tense (T) state. As expected, coupling of deoxy Hb (T-state) with HSAs yielded HbT–HSA 3 cluster having low O2 affinity. The mixing of HbR–HSA 3 and HbT–HSA 3 clusters conferred a tailor-made formulation of artificial O2 carrier with a desired O2 affinity (P 50).

A core-shell protein cluster comprising hemoglobin and human serum albumins, hemoglobin-albumin cluster (Hb-HSA 3), was designed and synthesized for use as an artificial O 2 carrier and red blood cell (RBC) substitute. For initial preclinical safety evaluation of the Hb-HSA 3 solution, we observed blood compatibility in vitro, physiological responses after exchange transfusion into rats and blood circulation lifetime in dogs. Dilution of human whole blood with Hb-HSA 3 showed an appropriate decrease in blood cell number, proportional to the mixing volume ratio. Time courses in the circulation parameters and blood gas parameters after 20% exchange transfusion with Hb-HSA 3 in anesthetized rats were almost identical to those observed in a sham group (without infusion) and an HSA group (with HSA administration) for 6 h. Serum biochemical tests of the withdrawn blood indicated safety of the protein cluster. Furthermore, fluorescent Hb-HSA 3 was infused into beagle dogs to assess blood retention. Fluorescence measurements of the blood samples enabled us to ascertain the cluster half-life within the intravascular space. Histopathologic inspections of the vital organs imply no abnormality in tissues. All these results indicate sufficient initial preclinical safety of Hb-HSA 3 as an alternative material for use in RBC transfusion.

There is no blood bank for pet animals. Consequently, veterinarians themselves must obtain “blood” for transfusion therapy. Among the blood components, serum albumin and red blood cells (RBCs) are particularly important to save lives. This paper reports the synthesis, structure, and properties of artificial blood for the exclusive use of dogs. First, recombinant canine serum albumin (rCSA) was produced using genetic engineering with Pichia yeast. The proteins showed identical features to those of the native CSA derived from canine plasma. Furthermore, we ascertained the crystal structure of rCSA at 3.2 Å resolution. Pure rCSA can be used widely for numerous clinical and pharmaceutical applications. Second, hemoglobin wrapped covalently with rCSA, hemoglobin–albumin cluster (Hb-rCSA3), was synthesized as an artificial O2-carrier for the RBC substitute. This cluster possesses satisfactorily negative surface net charge (pI = 4.7), which supports enfolding of the Hb core by rCSA shells. The anti-CSA antibody recognized the rCSA exterior quantitatively. The O2-binding affinity was high (P50 = 9 Torr) compared to that of the native Hb. The Hb-rCSA3 cluster is anticipated for use as an alternative material for RBC transfusion, and as an O2 therapeutic reagent that can be exploited in various veterinary medicine situations.

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