ABSTRACT
Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available "bridging molecules" containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants.
Subject(s)
Amines/chemistry , Click Chemistry/methods , Peptides/chemistry , Polyphosphates/chemistry , Biotinylation , Cyclooctanes/chemistry , Cystamine/chemistry , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Polyphosphates/isolation & purification , Sulfhydryl Compounds/chemistry , Temperature , Time FactorsABSTRACT
This illustrated review focuses on polyphosphate as a potent modulator of the plasma clotting cascade, with possible roles in hemostasis, thrombosis, and inflammation. Polyphosphates are highly anionic, linear polymers of inorganic phosphates that are widespread throughout biology. Infectious microorganisms accumulate polyphosphates with widely varying polymer lengths (from a few phosphates to over a thousand phosphates long), while activated human platelets secrete polyphosphate with a very narrow size distribution (about 60-100 phosphates long). Work from our lab and others has shown that long-chain polyphosphate is a potent trigger of clotting via the contact pathway, while polyphosphate of the size secreted by platelets accelerates factor V activation, blocks the anticoagulant activity of tissue factor pathway inhibitor, promotes factor XI activation by thrombin, and makes fibrin fibrils thicker and more resistant to fibrinolysis. Polyphosphate also modulates inflammation by triggering bradykinin release, inhibiting the complement system, and modulating endothelial function. Polyphosphate and nucleic acids have similar physical properties and both will trigger the contact pathway-although polyphosphate is orders of magnitude more procoagulant than either DNA or RNA. Important caveats in these studies include observations that nucleic acids and polyphosphate may co-purify, and that these preparations can be contaminated with highly procoagulant microparticles if silica-based purification methods are employed. Polyphosphate has received attention as a possible therapeutic, with some recent studies exploring the use of polyphosphate in a variety of formulations to control bleeding. Other studies are investigating treatments that block polyphosphate function as novel antithrombotics with the possibility of reduced bleeding side effects.
Subject(s)
Artifacts , Blood Coagulation Tests , Blood Coagulation/drug effects , DNA/isolation & purification , Polyphosphates/isolation & purification , Reagent Kits, Diagnostic , Silica Gel/pharmacology , Cell Fractionation/instrumentation , Cell Fractionation/methods , Centrifugation , Chloroform , DNA/pharmacology , Extracellular Traps/chemistry , HEK293 Cells , Humans , Phenol , Silica Gel/analysis , SolventsABSTRACT
Nanodiscs are monodisperse, self-assembled discoidal particles that consist of a lipid bilayer encircled by membrane scaffold proteins (MSP). Nanodiscs have been used to solubilize membrane proteins for structural and functional studies and deliver therapeutic phospholipids. Herein, we report on tetramethylrhodamine (TMR) tagged nanodiscs that solubilize lipophilic MR contrast agents for generation of multimodal nanoparticles for cellular imaging. We incorporate both multimeric and monomeric Gd(III)-based contrast agents into nanodiscs and show that particles containing the monomeric agent (ND2) label cells with high efficiency and generate significant image contrast at 7 T compared to nanodiscs containing the multimeric agent (ND1) and Prohance, a clinically approved contrast agent.
Subject(s)
Magnetic Resonance Imaging/methods , Multimodal Imaging/methods , Nanostructures/chemistry , Optical Imaging/methods , Contrast Media/chemistry , Gadolinium/chemistry , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , MCF-7 Cells , Models, Molecular , Molecular Conformation , Rhodamines/chemistry , SolubilityABSTRACT
Inorganic polyphosphates, linear polymers of orthophosphate, occur naturally throughout biology and have many industrial applications. Their biodegradable nature makes them attractive for a multitude of uses, and it would be important to understand how polyphosphates are turned over enzymatically. Studies of inorganic polyphosphatases are, however, hampered by the lack of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. We now report chromogenic and fluorogenic polyphosphate substrates that permit spectrophotometric monitoring of polyphosphate hydrolysis and allow for high-throughput analyses of both endopolyphosphatase and exopolyphosphatase activities, depending on assay configuration. These substrates contain 4-nitrophenol or 4-methylumbelliferone moieties that are covalently attached to the terminal phosphates of polyphosphate via phosphoester linkages formed during reactions mediated by EDAC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide). This report identifies Nudt2 as an inorganic polyphosphatase and also adds to the known coupling chemistry for polyphosphates, permitting facile covalent linkage of alcohols with the terminal phosphates of inorganic polyphosphate.