William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, United States of America.
PLoS One. 2022 Feb 16;17(2):e0263782. doi: 10.1371/journal.pone.0263782. eCollection 2022.
Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-[Formula: see text], and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.
高铁血红蛋白（metHb）是由亚铁血红蛋白（HbFe2+，Hb）氧化为高铁血红蛋白（HbFe3+，metHb）引起的，后者无法结合气态配体，如氧气（O2）和一氧化碳（CO），并且与一氧化氮（NO）的结合速度明显慢于 Hb。因此，与无细胞 Hb 相比，metHb 的一氧化氮清除率极低，因此不会在体内引起血管收缩和高血压，但会导致氧化组织损伤，这表明当研究无细胞 Hb 的毒性时，metHb 可作为对照材料使用。因此，本工作的目标是开发一种新的制造方法，以生产适合放大规模的 metHb。在这项研究中，首先进行了小规模（例如 1 mL 反应体积）筛选实验，以确定 Hb 与氧化剂过氧化氢（H2O2）或亚硝酸钠（NaNO2）的最佳摩尔比，以实现将 Hb 转化为 metHb 的最高转化率。采用光谱解卷积程序来确定在氧化反应过程中溶液中各种物质（半高铁血红素、metHb、氧合 Hb、metHb-[Formula: see text]和 NaNO2）的摩尔分数。通过该分析，确定了 Hb:NaNO2（以血红素为基础）的最佳摩尔比为 1:1 或 1:5，这可使 Hb 转化为 metHb 的转化率最高，同时副产物的量最小。因此，为了缩短反应时间，选择 1:5 的摩尔比用于牛 metHb（metbHb）和人 metHb（methHb）的大规模（即 1.5 L 反应体积）合成。然后对 metHb 的生物物理特性进行了表征，以阐明使用合成的 metHb 作为非 O2 携带对照材料的潜力。发现 metHb 的结合动力学与 Hb 相似。此外，合成的 metHb 在磷酸盐缓冲盐水（PBS，50 mM，pH 7.4）中在 4°C 下稳定约一周，表明该材料的稳定性高。
