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

• Published in: ACS omega Vol. 4; no. 2; pp. 3228 - 3233
• Main Authors: Funaki, Ryosuke, Kashima, Tomonori, Okamoto, Wataru, Sakata, Sho, Morita, Yoshitsugu, Sakata, Masayo, Komatsu, Teruyuki
• Format: Journal Article
• Language: English
• Published: American Chemical Society 28.02.2019
• ISSN: 2470-1343; 2470-1343
• DOI: 10.1021/acsomega.8b03474

The protein cluster comprising a bovine hemoglobin (Hb) wrapped covalently by human serum albumins (HSAs), Hb–HSA m 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–HSA m 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–HSA m 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 chromogenic limulus amebocyte lysate assay of lipopolysaccharides in the protein cluster solution.