Oxygen therapeutics are being developed for a variety of applications in transfusion medicine. In order to reduce the side effects (vasoconstriction, systemic hypertension, and oxidative tissue injury) associated with previous generations of oxygen therapeutics, new strategies are focused on increasing the molecular diameter of hemoglobin (Hb) obtained from mammalian sources via polymerization and encapsulation. Another approach toward oxygen therapeutic design has centered on using naturally occurring large molecular diameter Hbs [i.e., erythrocruorins (Ecs)] derived from annelid sources. Therefore, the goal of this study was to purify Ec from the terrestrial worm Lumbricus terrestris for diverse oxygen therapeutic applications. Tangential flow filtration was used as a scalable protein purification platform to obtain a >99% pure Lumbricus terrestris Ec (LtEc) product, which was confirmed by size exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In vitro characterization concluded that the ultrapure LtEc product had oxygen equilibrium properties similar to those of human red blood cells and a lower rate of auto-oxidation compared to human Hb, both of which should enable efficient oxygen transport under physiological conditions. In vivo evaluation concluded that the ultrapure product had positive effects on the microcirculation sustaining functional capillary density compared to a less pure product (∼86% purity). In summary, we purified an LtEc product with favorable biophysical properties that performed well in an animal model using a reliable and scalable purification platform to eliminate undesirable proteins.

中文翻译：

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用于多种氧气治疗应用的陆蠹巨血红蛋白的纯化

氧气疗法正在开发用于输血医学中的各种应用。为了减少与前几代氧疗法相关的副作用（血管收缩、系统性高血压和氧化性组织损伤），新的策略集中在增加通过聚合和封装从哺乳动物来源获得的血红蛋白 (Hb) 的分子直径。另一种氧治疗设计的方法集中在使用源自环节动物来源的天然存在的大分子直径 Hbs [即红红素 (Ecs)]。因此，本研究的目的是从陆生蠕虫Lumbricus terrestris 中纯化 Ec。用于各种氧气治疗应用。切向流过滤被用作可扩展的蛋白质纯化平台，以获得纯度 > 99% 的陆蠹 Ec (LtEc) 产品，这通过尺寸排阻高效液相色谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析得到证实。体外表征得出的结论是，超纯 LtEc 产品具有与人类红细胞相似的氧平衡特性，并且与人类 Hb 相比，自氧化速率较低，这两者都应该能够在生理条件下实现有效的氧传输。体内评估得出的结论是，与纯度较低的产品（~86% 纯度）相比，超纯产品对维持功能性毛细血管密度的微循环具有积极影响。总之，我们纯化了一种具有良好生物物理特性的 LtEc 产品，该产品使用可靠且可扩展的纯化平台来消除不需要的蛋白质，该产品在动物模型中表现良好。