A core-shell ensemble of bovine hemoglobin (Hb) and human serum albumin (HSA) is an artificial O-2 carrier as a red blood cell substitute. This protein particle is created by covalent wrapping of a carbonyl Hb with HSAs: Hb(R)-HSA(3) cluster, where Hb(R) signifies the use of carbonyl Hb (relaxed (R) state conformation) as a starting material. The Hb(R)-HSA(3) cluster exhibits high O-2 affinity and low cooperativity. Analysis of the quaternary structure of the central Hb(R) in the cluster revealed that its high O-2 affinity is attributed to the physically immobile Hb(R) nucleus. Circular dichroism and UV-vis absorption spectroscopy showed that the structure of deoxy Hb(R) core closely resembles the R-state. The crystal structure of Lys-modified carbonyl Hb(R) was superimposed on that of carbonyl Hb. These results imply that chemical modifications of the surface Lys groups and Cys-93(beta) 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 Hb(T)-HSA(3) cluster having low O-2 affinity. The mixing of Hb(R)- HSA(3) and Hb(T)-HSA(3) clusters conferred a tailor-made formulation of artificial O-2 carrier with a desired O-2 affinity (P-50).