Josić D, Schwinn H, Strancar A, Podgornik A, Barut M, Lim Y P, Vodopivec M
Octapharma Pharmazeutika Produktionsges, mbH, Wien, Austria.
J Chromatogr A. 1998 Apr 17;803(1-2):61-71. doi: 10.1016/s0021-9673(97)01282-x.
Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase i.v., which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate-polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.
将不同的高分子质量配体固定在紧密的多孔分离单元上，并用于亲和色谱。在后续实验中，固定不同的酶，并用于转化低分子质量和高分子质量的底物。固定有伴刀豆球蛋白A（ConA）的圆盘或试管用作凝集素亲和色谱的模型系统。使用葡萄糖氧化酶作为标准蛋白来测试ConA单元。随后，使用固定有ConA的单元分离大鼠肝脏质膜中的糖蛋白。在实验中用作模型蛋白的二肽基肽酶IV在单一步骤中富集约40倍，产率超过90%。结果仅比将ConA固定在块状载体上时略好。重要的改进在于将分离时间缩短至仅1小时。在圆盘上进行了关于抗凝血因子VIII（FVIII）单克隆抗体分离的实验，将阴离子交换色谱和蛋白A亲和色谱结合作为多维色谱的模型。来自小鼠腹水的IgG（结合到蛋白A圆盘上）和伴随蛋白（结合到阴离子交换圆盘上）都被阻滞并分别洗脱。使用固定化的转化酶和葡萄糖氧化酶（GOX）转化相应的低分子质量底物蔗糖和葡萄糖。结果表明，固定化酶的量和底物浓度决定了转化程度，而实验中使用的流速则完全没有影响。用GOX研究了固定化化学的影响。以ConA作为间隔物的间接固定被证明是最佳选择。将胰蛋白酶固定在圆盘上，高分子质量的底物在流通中被消化。为了实现最佳消化，蛋白质在应用前必须在用于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的缓冲液中变性。与低分子质量底物的转化不同，流速在高分子质量底物的转化中起着重要作用。流速越低，消化程度越高。
