Quaternary Structure Analysis of a Hemoglobin Core in Hemoglobin–Albumin Cluster

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: Hb^R–HSA₃ cluster, where Hb^R signifies the use of carbonyl Hb (relaxed (R) state conformation) as a starting material. The Hb^R–HSA₃ cluster exhibits high O₂ affinity and low cooperativity. Analysis of the quaternary structure of the central Hb^R in the cluster revealed that its high O₂ 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(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 Hb^T–HSA₃ cluster having low O₂ affinity. The mixing of Hb^R–HSA₃ and Hb^T–HSA₃ clusters conferred a tailor-made formulation of artificial O₂ carrier with a desired O₂ affinity (P₅₀).