I have purified a protein which suppose to adenylate amino acid consuming ATP and releasing AMP and PPi, so to test the protein activity I have to measure the released PPi in the solution. Can someone tell the test or kit can be used to do that?
Many thanks
I'm not sure how to measure PPi, but why don't you just measure the breakdown of ATP? Luciferase based measurements (such as ATP-Lite from Promega) are very sensitive and easy to do.
John
Look up the Fiske-Subbarow method. It is pretty old, but I have used this one and it works to determine orthophosphate. But first you would need diphosphatase to work on your PPi. Which is still pretty easy to do.
Two suggestions, each has worked well in my lab in the past. First is the EnzChek Pyrophosphate Assay Kit from Life Technologies. Pretty straightforward and accurate. Much cheaper though is the malachite green assay described in the following: Geladopoulos, T.P., T.G. Sotiroudis, and A. Evangelopoulos, A malachite green colorimetric assay for protein phosphatase activity. . Analytical Biochemistry, 1991.192: p. 112-116.
All the best - David
Long time ago I used to use the so-called coupled enzyme assay for detecting "ATPase activity"; means measuring ATP consumption. In this assay the ATP concentration is kept constant. Here you can find the assay in detail but the essence is as follows: "...The coupled enzyme ATPase assay is based on the conversion of phosphoenolpyruvate (PEP) to pyruvate by pyruvate kinase (PK) coupled to the conversion of pyruvate to lactate by lactate dehydrogenase (LDH). The latter step requires NADH which is oxidized to NAD+. NADH absorbs strongly at 340 nm but NAD+ does not, enabling the utilization of NADH to be followed by monitoring absorbance at 340 nm. The decrease in OD340 can be converted into ATPase activity where 1 molecule of NADH oxidized to NAD+ corresponds to the production of 1 molecule of ADP by the motor ATPase."
Sorry; it is the link: http://labs.cellbio.duke.edu/kinesin/Methods/ATPase_assay.html
... or this web page: http://openwetware.org/wiki/Sauer:ATPase_assays
You can use an assay of pyrophosphate I developed many years ago. It was later used as step in pyrosequencing. It is based on firefly luciferase and ATP sulfurylase. As you have ATP present in you reaction mixture you have to stop the reaction after some incubation time, degrade remaining ATP to ADP and thereafter convert pyrophosphate in the presence of APS and ATP sulfurylase back to ATP. BioThema has a suitable luciferin-luciferase reagent for you.
Sorry Hadi, my comment was not correct; it can be used only for detecting ATP hydrolysis to ADP + Pi. However, you have a reaction of ATP -> AMP + PPi. I was inattentive. I think Tomasz made the same mistake; Fiske-Subborow method works for Pi not for PPi.
So the right answer is with Jonathan and David; the luciferin-luciferase system is a right method to detect ATP consumption. However, there are many other assay kits on market which work fine.
@ Alajos: I told that you need to couple it with pirophosphatase reaction,)
I wonder if you could try HPLC to separate and quantitate it through standard curves using synthetic pyrophosphate, ATP and ADP?
Well as Adam points out that HPLC might help. But I would use the indirect method, that is measure the sdenylated aminoacids using quantitative LCMS instead of the PPi or ADP or AMP etc.That will be more accurate than any of the phosphate measurement results. You beleive me that when you will go for statistical evaluation of your data, adenylated amino acid quantification will prove to be very beneficial.
I think you could use gamma 32P radioactive ATP in reaction, and than run TLC ( very old method)/ You will be ble to mesure % of ATP hydrolysis. Look it up by google.
I have not done it but a simple way to address this need will be to use inorganic pyrophpsphatase enzyme to convert pyrophosphate into inorganic phosphate and then to estimate the phosphate according to Fiske-Subbarao method.
You will have to standardize a protocol for this.
hope this will help.
Good luck
Instead of measuring PPi you could measure AMP using three coupled enzymes: myokinase + pyruvate kinase + lactate dehydrogenase. Based on these enzymes you could designed a discontinuos dead point method, or a kinetic continuous one.
PPI is simple to measure at low concentrations but you have to degrade ATP Before the assay described in the attached paper.
Arne
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