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

Abstract

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).

Hemoglobin(βC93A)–Albumin Cluster: Mutation of Cysteine‐β93 to Alanine Allows Moderate Reduction of O2 Affinity by Inositol Hexaphosphate

“…As reported previously, the Hb‐HSA 3 cluster displayed low P 50 and n values ( P 50 : 8 torr, n= 1.4), relative to those of native Hb ( P 50 =12 torr, n= 2.4; Table ) . The reductions are attributed to chemical modifications of Cys‐β93 and surface Lys groups of R‐state Hb (raw material) by SMP . In particular, the covalent binding of bulky SMP to the Cys‐β93 residue interrupts the formation of the His‐β146 ⋅⋅⋅ Asp‐β94 salt bridge and destabilizes the T state (Figure B).…”

“…The groups of Rosa and Ho reported that capping of Cys‐β93 of Hb with N ‐ethylmaleimide or other sulfhydryl reagents also increased the O 2 affinity and weakened the IHP effect. We previously reported the synthesis of a core–shell‐structured protein cluster comprising of Hb and human serum albumin (HSA), the Hb‐HSA 3 cluster, as a promising HBOC . The Hb‐HSA 3 cluster has shown initial preclinical safety and long circulation persistence in the bloodstream .…”

“…The Hb‐HSA 3 cluster has shown initial preclinical safety and long circulation persistence in the bloodstream . We reasoned that the binding of a bulky crosslinker, α‐succinimidyl‐ω‐maleimide, to Cys‐β93 and surface Lys residues of Hb inhibited movement of the quaternary structure from the relaxed (R) state to the T state . Thereby, the Hb‐HSA 3 cluster shows 1) higher O 2 affinity and lower cooperativity than those of native Hb, and 2) no IHP effect.…”

“…By contrast, the Soret band absorption of the deoxy rHb(βC93A)‐HSA 3 cluster ( λ max =430 nm) was low. The weak Soret band suggested that the central rHb(βC93A) remained near the R state, even if fully deoxygenated …”

“…In particular, the covalent binding of bulky SMP to the Cys‐β93 residue interrupts the formation of the His‐β146 ⋅⋅⋅ Asp‐β94 salt bridge and destabilizes the T state (Figure B). Thus, quaternary structure conversion from the R to T state was affected . The Hb core is physically immobile near the R state, which affords a high O 2 affinity.…”