Covalent Core-Shell Architecture of Hemoglobin and Human Serum Albumin as an Artificial O2 Carrier

Covalent core-shell structured protein clusters of hemoglobin (Hb) and human serum albumin (HSA) (HbX-HSA(m)) (m = 2, 3) with novel physiological properties were generated by linkage of Hb surface lysins to HSA cysteine-34 via an alpha-succinimidyl-epsilon-maleimide cross-linker (X: 1 or 2). The isoelectric points of HbX-HSA(m) (pI = 5.0-5.2) were markedly lower than that of Mb and almost identical to that of HSA. AFM and TEM measurements revealed a triangular Hb1-HSA(3) cluster in aqueous medium. The complete 3D structure of Hb1-HSA(3) based on TEM data was reconstructed, revealing two possible conformer variants. All HbX-HSA(m) clusters showed a moderately higher O-2 affinity than the native Hb. Furthermore, the exterior HSA units possess a remarkable ability to bind lumiflavin (LF). The addition of NADH to an aqueous solution of the met-Hb2-(HSA-LF)(3) cluster reduced the inactive ferric Mb center to the functional ferrous Hb. This O-2-carrying hemoprotein cluster with strongly negative surface net charge, high O-2 affinity, and NADH-dependent reductase unit can support a new generation of molecular architecture for red blood cell substitutes.