Core–shell clusters of human haemoglobin A and human serum albumin: artificial O2-carriers having various O2-affinities

Abstract

This report describes the synthesis, structure, and O₂-binding properties of core–shell clusters composed of human haemoglobin A (HbA) in the centre and human serum albumin (HSA) at the periphery (HbA–HSA_m) as potential O₂-carriers designed as red blood cell (RBC) substitutes. Protein clusters were prepared by covalent linkages between HbA lysins and HSA cysteine-34 using a heterobifunctional cross-linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Major products (m = 2, 3, 4) were isolated using gel filtration chromatography. The low isoelectric points (pI = 5.0–5.2) of the clusters were almost identical to that of HSA, proving the wrapping of HbA by negatively charged HSA. The 3D reconstruction of HbA–HSA₃ based on transmission electron microscopy images revealed a complete triangular structure. The proposed geometries enabled us to assign a possible spatial arrangement of the HbA centre and HSA periphery. The HbA–HSA_m clusters showed higher O₂-affinities (P₅₀ = 8–9 Torr) than the native HbA. The clusters prepared under N₂ atmosphere showed a low O₂-affinity (P₅₀ = 26 Torr) resembling those of human RBC. Moreover, the cluster containing an αα-cross-linked HbA with bis(3,5-dibromosalicyl)fumarate showed a markedly low O₂-affinity (P₅₀ = 35 Torr). These HbA–HSA_m clusters with various O₂-affinities can support a new generation of RBC substitutes, which can be better tuned to play a role in O₂ transport.