Cross-Linked Hemoglobin-Albumin Cluster as an Artificial O_2

摘要

The decrease in blood donor population by the low birthrate and aging causes serious problem to bring a lack of blood transfusion. Under such social situation, the great current interest in synthesizing red blood cell (RBC) substitutes continues to grow. Hemoglobin (Hb)-based O_2 carriers of several kinds have been extensively developed, such as intramolecularly cross-linked Hb, polymerized Hb, and poly(ethylene- glycol)-conjugated Hb. However, none satisfies all requirements for use as an RBC substitute. A common side effect is mild hypertension resulting from NO scavenging by Hb diffused into extra vascular space. Recently, we have prepared core-shell structured hemoglobin-albumin cluster (Hb-HSA_3) by conjugation of Hb with human serum albumin (HSA) as a new type of artificial O_2 carrier. In this paper, we report for the first time the synthesis and O_2 binding property of more stable and robust protein cluster having intramolecularly cross-linked Hb as a core, ββHb-HSA_3 (Fig. 1). The ββHb-HSA_3 was synthesized by covalent wrapping ββHb with HSA. Initially, ββHb was prepared by cross-linking of two thiol groups in cysteine-93(β_1 and β_2) using 1,11-bis-(maleimido)triethylene glycol. Then, the obtained ββHb was wrapped covalently with HSA by linkage of Hb surface lysines to HSA cysteine-34 via succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate. Excess HSA was removed by gel filtration chromatography. HPLC and Native-PAGE measurements of the resultant product revealed that major component of the cluster was ββHb-HSA_3 heterotetramer. The isoelectric point (pI=5.1) was very close to the values of HSA and Hb-HSA_3. The UV-vis. absorption spectral pattern of ββHb-HSA_3 in response to N_2, O_2, CO were fundamentally identical to those of Hb and Hb-HSA_3. The O_2 affinity (P_(50)=6 Torr) was slightly higher than that of Hb-HSA_3. Consequently, ββHb-HSA_3 is expected to be useful as a unique artificial O_2 carrier having a long-term circulation in the bloodstream without subunit dissociation of the core Hb.