Kosík Ondrej, Farkas Vladimír
Institute of Chemistry, Center of Excellence GLYCOBIOS, Slovak Academy of Sciences, Dúbravská cesta 9, 84538 Bratislava, Slovakia.
Anal Biochem. 2008 Apr 15;375(2):232-6. doi: 10.1016/j.ab.2007.11.025. Epub 2007 Nov 28.
Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH(3) at 80 degrees C for 2-4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.
已证明，用磺基罗丹明荧光标记的木葡聚糖寡糖是评估植物木葡聚糖内转糖基酶/水解酶（XTH；EC 2.4.1.207）转糖基活性的一种有价值的工具。在此，我们描述一种制备它们的简单快速方法。相应地，起始的木葡聚糖衍生寡糖在第一步中通过与乙酸铵和NaCNBH(3)在80℃孵育2 - 4小时转化为其相应的1 - 氨基 - 1 - 脱氧醛糖醇（糖胺），在第二步中，糖胺与丽丝胺罗丹明B磺酰氯反应以获得寡糖糖胺的荧光标记衍生物。所有操作均在单个离心管中进行，并且基于它们在有机溶剂中的不同溶解度分离各个反应步骤的产物。使用所述方案，整个过程可在不到24小时内完成。由此获得的磺基罗丹明标记的木葡聚糖寡糖被证明适合作为用于XTH转糖基活性灵敏荧光测定的底物。
