Schrader Silke, Tiralongo Evelin, Paris Gastón, Yoshino Teruo, Schauer Roland
Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, 24098, Kiel, Germany.
Anal Biochem. 2003 Nov 15;322(2):139-47. doi: 10.1016/j.ab.2003.07.016.
Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.
转唾液酸酶（E.C. 3.2.1.18）催化将优先为α2,3连接的唾液酸转移至另一种聚糖或糖缀合物，形成与半乳糖或N-乙酰半乳糖胺的新α2,3连接。在此，我们描述了一种非放射性96孔板荧光试验，分别使用唾液乳糖和4-甲基伞形酮基-β-D-半乳糖苷作为供体和受体底物，以高灵敏度、特异性和可重复性监测转唾液酸酶活性。使用来自刚果锥虫的转唾液酸酶对测定条件进行了优化，并用来自克氏锥虫的重组转唾液酸酶证实了其普遍适用性。使用该方法，可以快速、可靠地检测大量样品，例如在酶纯化和单克隆抗体制备过程中监测转唾液酸酶、进行酶的特性鉴定以及识别潜在的底物和抑制剂。本文报道的转唾液酸酶测定法能够检测低毫微单位范围内的转唾液酸酶活性，可能是在各种生物系统中寻找更多转唾液酸酶的有价值工具。
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