TNP-ATP

TNP-ATP is a fluorescent molecule that is able to determine whether or not a protein binds to ATP, and the constants associated with that binding. It is primarily used in fluorescence spectroscopy, but is also very useful as an acceptor molecule in FRET, and as a fluorescent probe in fluorescence microscopy and X-ray crystallography.

Constituent parts

TNP refers to the chemical compound 2,4,6-trinitrophenol, also known as Picric acid. It is a primary constituent of many unexploded landmines, and is a cousin to TNT, but less stable. It is recognized as an environmental contaminant and is toxic to many organisms. It is still commonly used in the manufacture of fireworks, explosives, and rocket fuels, as well as in leather, pharmaceutical, and dye industries.

ATP is an essential mediator of life. It is used to overcome unfavorable energy barriers to initiate and fuel chemical reactions. It is also used to drive biological machinery and regulate a number of processes via protein-phosphorylation. However, the proteins that bind ATP for both regulation and enzymatic reactions are very diverse—many yet undiscovered—and for many proteins their relationship to ATP in terms of number of binding sites, binding constants, and dissociation constants remain unclear.

TNP-ATP

Conjugating TNP to ATP renders this nucleotide triphosphate fluorescent and colored whilst allowing it to retain its biological activity. TNP-ATP is thus a fluorescent analog of ATP. This conjugation is very useful in providing information about interactions between ATP and an ATP-binding protein because TNP-ATP interacts with proteins and enzymes as a substitute for its parent nucleotide, and has a strong binding affinity for most systems that require ATP.

TNP is excited at a wavelength of 408 and 470 nm, and fluoresces in the 530–560 nm range. This is a very useful range of excitation because it is far from where proteins or nucleotides absorb. When TNP-ATP is in water or other aqueous solutions, this emission is very weak. However, once TNP-ATP binds to a protein, there is a dramatic increase in fluorescent intensity. This property enables researchers to study various proteins’ binding interaction with ATP. Thus, with enhanced fluorescence, it can be seen whether or not a protein binds to ATP.

When TNP-ATP in water is excited at 410 nm, TNP-ATP shows a single fluorescence maximum at 561 nm. This maximum shifts as the fluid's viscosity changes. For example, in N,N-dimethylformamide, instead of having its maxima at 561 nm as in water, the maxima is instead at 533 nm.

Binding to a protein will also change the wavelength of maximal emission, as well as a change in fluorescent intensity. For example, binding to the chemotaxis protein CheA indicates a severalfold enhancement of fluorescence intensity and a blue-shift in wavelength of the maximal emission.

Using this TNP nucleotide analog has been shown in many instances to be superior to traditional radionucleotide-labelling based techniques. The health concerns and the cost associated with the use of radioactive isotopes makes TNP-ATP an attractive alternative.

The first fluorescent ribose-modified ATP is 2’,3’-O-(2,4,7-trinitrocyclohexadienylidene) adenosine 5’triphosphate (TNP-ATP), and was introduced in 1973 by Hiratsuka and Uchida. TNP-ATP was originally synthesized to investigate the ATP binding site of myosin ATPase. Reports of TNP-ATP’s success in the investigation of this motor protein extended TNP-ATP’s use to other proteins and enzymes. TNP-ATP has now been used as a spectroscopic probe for numerous proteins suspected to have ATP interactions. These include several protein kinases, ATPases, myosin, and other nucleotide binding proteins. Over the past twenty years, there have been hundreds of papers describing TNP-ATP’s use and applications. Many applications involving this fluorescently labeled nucleotide have helped to clarify structure-function relationships of many ATP-requiring proteins and enzymes. There have also been a growing number of papers that display TNP-ATP use as a means of assessing the ATP-binding capacity of various mutant proteins.

Preparation

Preparing TNP-ATP is a one-step synthesis that is relatively safe and easy. Adenosine’s ribose moiety can be trinitrophenylated by 2,4,6-trinitrobenzene-1-sulfonate (TNBS). The resulting compound assumes a bright orange color and has visible absorption characteristics, as is characteristic of a Meiseinheimer spiro complex compound linking.

To see the exact method of preparion, please refer to T. Hiratsuka's and K. Uchida's paper "Preparation and Properties of 2'(r 3')-O(2,4,6-trinitrophenyl) Adenosine 5'-triphosphate, an Analog of Adenosine Triphosphate," found in the reference section.

To revert TNP-ATP back to its constituent parts, or in other words to hydrolyze TNP-ATP to give equilmolar amounts of picric acid (TNP) and ATP, TNP-ATP should be treated with 1 M HCl at 100 degrees Celsius for 1.5 hours. This is because if TNP-ATP is acidified under mild conditions, it results in the opening of the dioxolane ring attached to the 2’-oxygen, leaving a 3’O-TNP derivative as the only product.

Storage

TNP-ATP should be stored at −20 degrees Celsius, in the dark, and used under minimal lighting conditions. When in solution, TNP-ATP has a shelf-life of about 30 days.