Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters

Abstract

A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

Fig 1. Schematic illustrations of hemoglobin-albumin clusters.

(A) Molecular structures of Hb-HSA₃, Hb-HSA₄, and XLHb-HSA₃. (B) Structural model of intramolecularly crosslinked Hb, in which sulfhydryl groups of Cys-93(β) residues (distance: 21.6 Å) are connected by BMTEG (molecular length: 18.3 Å, shown in a space-filling representation) [25,26].

Fig 2

SEC profiles of (A) Hb-HSA₄ and (B) XLHb-HSA₃. Dotted black lines are elution curves of the reaction mixture. Solid lines are elution curves of purified clusters.

Fig 3. Diameters and IEF patterns of hemoglobin-albumin clusters.

(A) Hydrodynamic diameters of Hb-HSA₃, Hb-HSA₄, and XLHb-HSA₃ measured using DLS. (B) IEF patterns of Hb-HSA₃, Hb-HSA₄, and XLHb-HSA₃.

Fig 4. Blood retention of hemoglobin-albumin clusters.

Relative plasma concentrations of ¹²⁵I-labeled Hb-HSA₃, Hb-HSA₄, XLHb-HSA₃, and HSA after intravenous administration to rats. Each data point represents the mean ± SD (n = 6). **p < 0.01 vs. HSA.

Fig 5. Tissue distribution of hemoglobin-albumin clusters.

Tissue (vital organs) distribution of ¹²⁵I radioactivity (% of dose) (A) at 1 h and (B) at 24 h after intravenous administration of ¹²⁵I-labeled Hb-HSA₃, Hb-HSA₄, XLHb-HSA₃, and HSA to rats. Each bar shows the mean ± SD (n = 6). **p < 0.01 vs. HSA.

Fig 6. Urinary and fecal excretions.

Urinary and fecal excretions of ¹²⁵I radioactivity (% of dose) within 24 h after intravenous administration of ¹²⁵I-labeled Hb-HSA₃, Hb-HSA₄, XLHb-HSA₃, and HSA to rats. Each bar shows the mean ± SD (n = 6). *p < 0.05 vs. Hb-HSA₃, **p < 0.01 vs. Hb-HSA₄.