Division of Pure and Applied Biochemistry, Department of Chemistry, Lund University, Box 124, 22100 Lund, Sweden.
Division of Pure and Applied Biochemistry, Department of Chemistry, Lund University, Box 124, 22100 Lund, Sweden.
Talanta. 2019 Jul 1;199:27-31. doi: 10.1016/j.talanta.2019.01.125. Epub 2019 Feb 12.
Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mLresin for adult Hb (HbA) and fetal Hb (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA αY42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an αA19D-αA12D HbF mutant eluted earlier, at -2.7 column volumes compared to wildtype HbF.
设计一种稳健、高效且经济实用的血红蛋白 (Hb) 纯化策略是开发基于 Hb 的血液代用品的关键挑战之一。本研究旨在使用分子印迹聚合物 (MIP) 作为一种新型且高效的色谱树脂，来选择性地识别和纯化不同的 Hb 变体。结果表明，这里开发的 Hb-MIP 材料可直接从大肠杆菌粗提物或人体体液（如血浆和脑脊液）一步中选择性地识别和纯化各种 Hb。在 10%穿透时的动态结合容量约为 7.4mg/mL 树脂的成人 Hb（HbA）和胎儿 Hb（HbF）。这种色谱材料还可以识别与 Hb 蛋白表面氨基酸取代相关的变化。例如，当引入额外的赖氨酸残基时，HbA αY42K 突变体在 Hb-MIP 柱上的洗脱时间晚于野生型 HbA。蛋白表面上额外的负电荷，如天冬氨酸，减轻了蛋白与印迹聚合物之间的相互作用，因此与野生型 HbF 相比，αA19D-αA12D HbF 突变体的洗脱时间更早，在-2.7 个柱体积处洗脱。
