Tangential Flow Filtration of Hemoglobin
Tangential Flow Filtration of Hemoglobin
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DOI：
10.1002/btpr.119
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发表时间：
2009-01-01
影响因子：
2.9
通讯作者：
Harris, David R.
中科院分区：
文献类型：
--
作者：
Palmer, Andre F.;Sun, Guoyong;Harris, David R.
牛和人血红蛋白（分别为bHb和hHb）通过切向流过滤（TFF）在四个连续阶段从牛和人红细胞中纯化。TFF是一种通过中空纤维（HF）膜过滤从RBC中纯化Hb的快速简单方法。大部分Hb保留在第III阶段（100 kDa HF膜），高铁血红蛋白水平低于1%，bHb和hHb的最终浓度分别为318和300 mg/mL。纯化的Hb表现出比它们各自的RBC低得多的内毒素水平。最初通过SDS-PAGE评估Hb的纯度，并显示出阶段III截留物的微小杂质条带。根据测定的红细胞和纯化血红蛋白的氧-红细胞/血红蛋白平衡曲线，回归出氧亲和力（P-50）和协同系数（n）。这些结果表明，TFF产生的bHb和hHb的氧亲和力与文献中的值相当。LC-MS用于测量纯化Hb的α和β珠蛋白链的分子量。是否未出现杂质峰？纯化Hb的HPLC色谱图。对应于α和β珠蛋白链的分子离子的质量与α和β珠蛋白链的计算理论质量一致。综上所述，我们的结果表明，HPLC级Hb调用通过TFF产生。通常，该方法可以更广泛地应用于从任何来源的RBC中纯化Hb。这项工作是有意义的，因为它概述了一种简单的方法，用于产生Hb的合成和/或配制Hb基氧载体。(C)0 2008年美国化学工程师学会生物技术。程序：25：189-199，2009
Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P-50) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (alpha) and beta (beta) globin chains of purified Hb. No impurity peaks were present it? the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the alpha and beta globin chains agreed well with the calculated theoretical mass of the alpha- and beta- globin chains. Taken together, our results demonstrate that HPLC-grade Hb call be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers. (C)0 2008 American Institute of Chemical Engineers Biotechnol. Prog., 25: 189-199, 2009