Eliminating disulfide exchange during glutamyl endopeptidase digestion of native protein
Eliminating disulfide exchange during glutamyl endopeptidase digestion of native protein
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DOI：
10.1016/s0021-9673(99)01028-6
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发表时间：
1999-12-24
影响因子：
4.1
通讯作者：
Regnier, FE
中科院分区：
文献类型：
--
作者：
Dormady, SJ;Lei, JM;Regnier, FE
在文献中引用了许多使用固定化酶比自由溶液蛋白质消化的优点。本研究考察了天然蛋白水解过程中形成的二硫桥接二肽片段的水解速率和二硫键交换程度。谷氨酰内肽酶作为固定化酶和游离酶被用于这些研究。结果发现，固定化酶盒在停止流动模式下操作，胰岛素在2分钟内实现了广泛的水解。这比在自由溶液中看到的要快几个数量级。从易于使用和减少样本量到自动化潜力的其他优点也注意到固定化酶试剂盒。正常的自由溶液蛋白水解通常需要12-24小时，基于溶液中较低的酶与底物比。在这些冗长的自由溶液反应中注意到的一个令人不安的特征是倾向于形成二硫桥接肽产物。这可能会导致错误的结论，即样品中的二硫键不是天然蛋白质的二硫键。结果表明，固定化酶相对于自由溶液反应的优势将在制药工业中发挥重要作用，在制药工业中，重组蛋白的蛋白水解片段“指纹图谱”被用于确定结构。(C) 1999 Elsevier Science B.V.版权所有
Numerous advantages of using immobilized enzymes over free-solution protein digests have been cited in the literature. This investigation examines both the rate of hydrolysis and the extent of disulfide bond exchange in disulfide bridged dipeptide fragments formed during proteolysis of native protein. Gutamyl endopeptidase as both an immobilized enzyme and in free solution was used in these studies. It was found that extensive hydrolysis of insulin was achieved in 2 min with immobilized enzyme cartridges operated in the stopped-flow mode orders. This is orders of magnitude faster than was seen in free solution. Other advantages ranging from ease of use and reduction in sample size to the potential for automation were also noted with the immobilized enzyme cartridge. Normal free-solution proteolysis generally requires 12-24 h, based on the lower enzyme-to-substrate ratio in solution. A disturbing feature noted in these lengthy free-solution reactions was the tendency to form disulfide bridged peptide artifacts. This could lead to the erroneous conclusion that disulfide bonding in a sample was not that of the native protein. It is concluded that the advantage of immobilized enzymes over free-solution reactions will be most important in the pharmaceutical industry where proteolytic fragment "fingerprinting" of recombinant proteins is being used to confirm structure. (C) 1999 Elsevier Science B.V. All rights reserved.