Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography
Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography
复制标题
DOI：
10.1016/j.jchromb.2008.02.014
复制
发表时间：
2008-05-01
影响因子：
3
通讯作者：
Palmer, Andre F.
中科院分区：
文献类型：
--
作者：
Sun, Guoyong;Palmer, Andre F.
牛和人的血红蛋白(Hb)构成了许多不同类型的基于Hb的O(2)载体(HBOC)的基础，从化学修饰的HBS到颗粒包裹的HBS。因此，开发一种简便的制备超纯Hb的纯化方法对于可靠地合成和制备RBOCs是至关重要的。在这项工作中，我们描述了一种纯化牛和人Hb超纯溶液的简单工艺。将牛和人红细胞裂解，用阴离子交换层析法从裂解液中分离纯化Hb。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析Hb组分的初始纯度。将纯Hb组分(对应于SDS-PAGE凝胶上的单一条带)汇集在一起，通过LC-MS鉴定总体纯度和鉴定。LC-MS分析得到两个峰，与计算的Hb的α和β链的理论相对分子质量相对应。通过测定Hb的氧亲和力、协作性和高铁血红蛋白水平来评价Hb的活性。这些活跃度的衡量标准与文献中的值相当。综上所述，我们的结果表明，通过阴离子交换层析可以很容易地制备出超纯的Hb(电泳法和HPLC法)。总的来说，这种方法可以更广泛地应用于从任何来源的红细胞中提纯血红蛋白。这项工作意义重大，因为它概述了一种简单的方法来产生用于合成和/或配制HBOC的超纯Hb。(C)2008爱思唯尔B.V.保留所有权利。
Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of RBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs. (C) 2008 Elsevier B.V. All rights reserved.