Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity
Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity
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氟辅助金属螯合物亲和萃取技术分析蛋白激酶活性
DOI：
10.1016/j.talanta.2016.04.058
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发表时间：
2016
期刊：
影响因子：
6.1
通讯作者：
Hitoshi Nohta
中科院分区：
文献类型：
--
作者：
Tadashi Hayama;Ena Kiyokawa;Hideyuki Yoshida;Osamu Imakyure;Masatoshi Yamaguchi;Hitoshi Nohta
我们开发了一种基于氟亲和的提取方法用于测量蛋白激酶活性。在该方法中，荧光肽底物被磷酸化的蛋白激酶，并获得的磷酸肽被选择性地捕获与Fe（III）-固定的全氟烷基亚氨基二乙酸试剂通过金属螯合亲和技术。接着，利用氟亲和性的特异性（亲氟性），将捕获的磷酸肽选择性地提取到含氟溶剂混合物中，所述含氟溶剂混合物为十四碳正己烷和1H，1H，2 H，2 H-十三碳正辛醇（3：1，v/v）。相比之下，水相（非氟）中剩余的底物肽很容易通过荧光法测量。最后，可以通过测量荧光的减少来测定酶活性。应用羧四甲基罗丹明（塔姆拉）标记底物肽（kemptide）测定cAMP依赖性蛋白激酶（PKA）活性的方法，证明了该方法的可行性。
We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).