Phosphoramidate end labeling of inorganic polyphosphates: facile manipulation of polyphosphate for investigating and modulating its biological activities.
Phosphoramidate end labeling of inorganic polyphosphates: facile manipulation of polyphosphate for investigating and modulating its biological activities.
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DOI：
10.1021/bi1014437
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发表时间：
2010-11-16
期刊：
影响因子：
2.9
通讯作者：
Morrissey, James H.
中科院分区：
文献类型：
--
作者：
Choi, Sharon H.;Collins, Julie N. R.;Smith, Stephanie A.;Davis-Harrison, Rebecca L.;Rienstra, Chad M.;Morrissey, James H.
多磷酸盐是通过磷酸酐键连接的无机磷酸盐的线性聚合物，广泛存在于生物体中，并在生物学中发挥着多种作用，包括作为人类凝血系统的有效天然调节剂。然而，由于缺乏衍生化聚磷酸盐或将其固定到固体支持物上的方法，蛋白质-聚磷酸盐相互作用的研究受到阻碍。我们现在报道，EDAC（1-乙基-3-[3-二甲基氨基-丙基]碳二亚胺）通过稳定的氨基磷酸酯键，有效促进各种含伯胺的标记和探针与聚磷酸盐的末端磷酸盐共价连接。使用 31P NMR，我们证实了伯胺和聚磷酸盐之间 EDAC 介导的反应导致氨基磷酸酯与末端磷酸基团连接。我们证明多磷酸盐可以被生物素化、用荧光团标记并固定在固体支持物上；固定化多磷酸盐可轻松用于定量蛋白质结合亲和力；共价衍生化或固定化的聚磷酸盐保留其引发血液凝固的能力；并且用亚精胺衍生化多磷酸盐的末端可以保护其免受外切多磷酸酶降解。我们的研究结果基本上打开了修饰多磷酸盐的整个蛋白质化学武器库，这将极大地促进对其生物学作用的研究。
Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of protein-polyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-dimetyhlamino-propyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary amine-containing labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using 31P NMR, we confirmed that EDAC-mediated reactions between primary amines and polyphosphate results in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores and immobilized onto solid supports; that immobilized polyphosphate can be readily used to quantify protein binding affinities; that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting; and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles.
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DOI：
10.1111/j.1538-7836.2008.03104.x
发表时间：
2008-10
期刊：
Journal of thrombosis and haemostasis : JTH
影响因子：
--
作者：
Smith SA;Morrissey JH
通讯作者：
Morrissey JH
影响因子：
5.3
作者：
Kim, Donghee;Cavanaugh, Eric J.
通讯作者：
Cavanaugh, Eric J.
DOI：
10.1016/s0167-4838(01)00193-5
发表时间：
2001-06-11
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY
影响因子：
--
作者：
Lorenz, B;Schröder, HC
通讯作者：
Schröder, HC
影响因子：
9.7
作者：
Morita, K.;Doi, K.;Akagawa, Y.
通讯作者：
Akagawa, Y.
影响因子：
64.5
作者：
Müller F;Mutch NJ;Schenk WA;Smith SA;Esterl L;Spronk HM;Schmidbauer S;Gahl WA;Morrissey JH;Renné T
通讯作者：
Renné T