Hemoglobin–Albumin Clusters Prepared Using N-Succinimidyl 3-Maleimidopropionate as an Appropriate Cross-Linker

The protein cluster comprising a bovine hemoglobin (Hb) wrapped covalently by human serum albumins (HSAs), Hb–HSAm cluster (m = 3.0 ± 0.2), is a promising artificial O2 carrier as a red blood cell substitute. Evaluating the safety and efficacy of the Hb–HSAm cluster solution in preclinical and clinical tests necessitates enlargement of the preparation scale and establishment of a simple purification system. This paper describes details of the ideal synthesis of the Hb–HSAm cluster and its O2 binding property. Results show that (i) N-succinimidyl 3-maleimidopropionate (SMP) is an appropriate cross-linker and (ii) anion-exchange chromatography (AEC) is effective for purification. Soluble SMP allows the combination of Hb with HSA in a high protein concentration. Results demonstrate that AEC enables us to separate the cluster and HSA by increasing the ionic strength of the medium. Individual cluster components (Hb–HSA4, Hb–HSA3, Hb–HSA2, and Hb–HSA) showed equal O2 affinity. Furthermore, we conducted chromoge...