Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000, Belgrade, Serbia.
Appl Biochem Biotechnol. 2012 Mar;166(6):1491-506. doi: 10.1007/s12010-012-9543-9. Epub 2012 Jan 18.
In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.
在这项工作中，我们描述了一种基于逐渐溶血的优化程序，用于从牛屠宰场红细胞中在膜生物反应器中分离血红蛋白。设计了一种在中试规模下提供高产量血红蛋白（主要是氧合血红蛋白衍生物）及其与空红细胞膜（红细胞）分离的膜生物反应器系统。从溶血程度、红细胞悬浮液的血细胞比容值、细胞肿胀以及盐浓度降低引起的膜变形等方面评估了 10 种不同浓度的低渗介质。使用 35 mM 的 Na-磷酸盐/NaCl 缓冲液（pH 值为 7.2-7.4）进行有效的逐渐渗透溶血，溶血程度为 88%。在这些条件下，大多数细胞膜在相差显微镜下呈现正常红细胞的外观。SDS-PAGE 证实血红蛋白的纯度>80%。动力学研究表明，血红蛋白的最大浓度在 40 分钟后达到，但实现 83%回收率的过程循环持续了 90 分钟。评估了两个步骤的动力学，（1）在溶血过程中通过膜运输红细胞，以及（2）通过反应器过滤器的运输。
