Phosphoramidate End-labeling of Inorganic Polyphosphates: Facile Manipulation of Polyphosphate for Investigating and Modulating its Biological Activities

摘要

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.