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.

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Myoglobin combined with human serum albumin (Mb-HSA) can be produced using yeast Pichia pastoris as a host strain, with secretion into the culture medium. This Mb-HSA fusion protein possesses identical O 2 binding affinity to that of naked Mb. The Mb unit is reconstituted with a zinc(II) protoporphyrin IX, yielding (zinc substituted Mb)-HSA, ZnMb-HSA. The photophysical property and singlet O 2 generation ability of ZnMb-HSA are equivalent to those of ZnMb. In vitro cell experiments revealed that ZnMb-HSA acts as a superior photosensitizer for photodynamic cancer therapy. It is noteworthy that ZnMb-HSA shows long circulation lifetime in vivo.

A bovine hemoglobin (HbBv) or human adult hemoglobin (HbA) wrapped covalently by human serum albumins (HSAs), hemoglobin–albumin clusters (HbBv–HSA3 and HbA–HSA3), are artificial O2 carriers used as a red blood cell substitute. This article describes the physicochemical properties of the HbBv–HSA3 and HbA–HSA3 solutions, and their abilities to restore the systemic condition after resuscitation from hemorrhagic shock in anesthetized rats. The HbBv–HSA3 and HbA–HSA3, which have high colloid osmotic activity, showed equivalent solution characteristics and O2 binding parameters. Shock was induced by 50% blood withdrawal. Rats exhibited hypotension and significant metabolic acidosis. After 15 min, the rats were administered shed autologous blood (SAB), HbBv–HSA3, HbA–HSA3, or Ringer's lactate (RL) solution. Survival rates, circulation parameters, hematological parameters, and blood gas parameters were monitored during the hemorrhagic shock and for 6 h after administration. All rats in the SAB, HbBv–HSA3, and HbA–HSA3 groups survived for 6 h. The HbBv–HSA3 and HbA–HSA3 groups restored mean arterial pressure after the resuscitation. No remarkable difference was observed in the time courses of blood gas parameters in any resuscitated group except for the RL group. Serum biochemical tests showed increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the HbBv–HSA3 and HbA–HSA3 groups compared to the SAB group. Therefore, we observed other rats awakened after resuscitation with HbA–HSA3 for 7 days. The blood cell count, AST, and ALT recovered to the baseline values by 7 days. All the results implied that HbBv–HSA3 and HbA–HSA3 clusters provide restoration from hemorrhagic shock as an alternative material for SAB transfusion.

This paper describes the synthesis and O 2 binding properties of core− shell structured hemoglobin (Hb) nanoparticles (NPs), artificial O 2 carriers of five types, as designed for use as red blood cell (RBC) substitutes. Human adult Hbs were polymerized using α-succinimidyl-ω-maleimide and dithiothreitol in spheroidal shapes to create parent particles. Subsequent covalent wrapping of the sphere with human serum albumin (HSA) yielded 100 nm-diameter Hb nanoparticles (HbNPs). The HbNP showed higher O 2 affinity than that of RBC, but NPs prepared under a N 2 atmosphere exhibited low O 2 affinity. Entirely synthetic particles comprising recombinant human adult Hb and recombinant HSA were also fabricated. Using a recombinant Hb (rHb) variant in which Leu-β28 of the heme pocket had been replaced with Phe, we found somewhat low O 2 affinity of rHb(βL28F)NP. Particles made of stroma-free Hb (SFHb) containing natural antioxidant enzyme catalase (SFHbNP) formed a very stable O 2 complex, even in aqueous H 2 O 2 solution. The SFHbNP showed good blood compatibility and did not affect the blood cell component functionality. The circulation half-life of SFHbNP in rats was considerably longer than that of naked Hb. All results indicate these Hb-based NPs as useful alternative materials for RBC and as a useful O 2 therapeutic reagent in diverse medical scenarios.

Non-C -units terpenoids (norisoprenoids) with an acetonyl group are widely distributed in nature. However, studies on the biosynthesis of norisoprenoids are scarce. Now, the C norisoprenoid, (all-E)-farnesylfarnesylacetone, was identified from Bacillus spp. and it was elucidated for the first time that superoxide mediates the cleavage of menaquinones (vitamin K) to form norisoprenoids in saponification treatment. From in vivo experiments using gene-disrupted Bacillus subtilis strains targeted for enzymes responsible for menaquinone biosynthesis and for superoxide dismutase, it was suggested that the non-enzymatic cleavage (autoxidation) of menaquinone with superoxide resulted in norisoprenoid synthesis in Bacillus cells. Furthermore, the bioactive norisoprenoids, farnesylacetone and phytone, were produced in Bacillus cells by this novel synthesis system.

Recombinant human haemoglobin expressed in Pichia yeast was wrapped covalently with recombinant human serum albumins, yielding a core–shell structured rHbA(X)–rHSA3 cluster as an entirely synthetic O2 carrier used for a red blood cell substitute.

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