Hi there,

Instead of trying to dose Pi why don't you try to couple your arginine kinase activty assay with pyruvate kinase and LDH? It would allow you to follow the reaction by following the stoechiometric oxydization of NADH  at 340nm?

Hi Dominique, 

I was trying to to that, but as my intention is measuring inhibition of the enzyme I started having troubles since some of my inhibitor molecules are antioxidants, and some of them even have high UV absorption at 340nm.

If you do not separate the phospho-arginine from the ATP, heating the samples will definitely cause the release of inorganic phosphate from ATP.

In additon to the PK/LDH method mentioned by Dominique Liger, there are a variety of reagents available for detection of the ADP product of the arginine kinase reaction, such as ADP-Glo for luminescence plate readers. They are costly, however.

You could use HPLC to separate and quantify ADP from ATP, as long as the concentration is high enough.

Can you use radioisotopes in your lab? I'm thinking about using thin-layer chromatography to separate the products from the reagents, and radiometric quantitation of the products.

Do you have access to LC-MS equipment for direct detection of phosphoarginine?

Hi Edward, I am familiar with your work; I hope this could help you:

That approach you're using isn't reliable because both ADP and ATP have very labile phosphoryl groups, and when you boil the samples probably a considerable part of inorganic phosphate is released from these sources.

The method I will propose to you is time consuming, but I think, you can make it in your lab without problems of infrastructure:

Have think about the remaining concentration of ATP in the reaction? You could measure it by using the delta DO and the lambert-beer law and a Hexokinase-G6PDH coupled assay.

Then, you have to prepare the reactions of the arginine kinase with your inhibitors and pick up at several times aliquots of the reaction and freeze rapidly, maybe with liquid nitrogen. After that, you can measure the remaining ATP in the reaction of the thawed aliquots, it is not going to be a direct assay, It will be an indirect assay with discrete points that you have to plot in order to obtain the primary curves of substrate concentration vs time.

To do this, you have to be sure that the concentration of the ATP in the second reaction is under the concentration of the other substrates, specially the NADP+. This method is reliable if you go on it carefully.

In the second reaction you will avoid the problems of absorbance of the inhibitory compounds because they will be diluted, and, moreover you will measure the delta DO.    

ATP + arginine à ADP + arginine-phosphate

Remaining ATP + glucose àglucose-6-phosphate + NADP+ à 6-phosphogluconate + NADPH

I hope this could help you.

Best regards Edward

When using the method recommended by Angel Lobo of measuring ATP remaining after the reaction (substrate depletion), there are some important points to consider. The method requires that a substantial portion of the ATP gets consumed by the reaction, such as half. If the portion is too small, such as 10%, it is difficult to measure precisely.

When using up such a large portion of the substrate, you run the risk of not working under initial rate conditions. In other words, the reaction slows down because of the drop in the substrate concentration and the accumulation of the product (product inhibition). It is essential for steady-state kinetics and for accurately measuring inhibitor potency that you maintain initial rate conditions.

To avoid the reaction slowing down despite consuming so much of the substrate, you must set the substrate concentration to be much higher than its Km. This has the drawback that the potency of inhibitors that compete with that substrate will be reduced. For non-competitive inhibitors, this is not a problem.

Also remember that for each molecule of ATP consumed, a molecule of arginine is consumed. So you also have to think about how much arginine can be consumed before the reaction slows down, how much arginine must be added to keep that from happening, and how the arginine concentration will affect the potency of inhibitors that compete with arginine.

(By the way, for inhibitors that are uncompetitive with one of the substrates, a high concentration of the other substrate actually increases the inhibitor potency. So a high substrate concentration can work in your favor under the right circumstances.)

For the above reasons, it is usually preferable to measure the formation of one of the products, if possible, rather than consumption of one of the substrates.

Hi Adam Shapiro, that's right what you are taking into account. Thank you for clearing some concepts, I didn´t know about the relationship between substrate concentration and uncompetitive inhibitors. Very thank you

Hello Adam and Angel, since I've got no access to MS, HPLC, radioisotopes or fluorecence plate readers, then I'm pretty stucked. I guess I'd keep using PK/LDH and ignore inhibitors with UV absorbtion at 340nm (and hope the other inhibitors don't mess with the coupled enzymes).

I'll keeo searching for another method (I found some assays designed for AK activity meassurement where molibdate and ascorbic acid is used, but again they rely on acid/heat hidrolization of P-arginine so I guess it's the same problemn as using malachite green)

Thanks, if I find my way I'll tell you.

Edward, Here is a reference that explains a simple way to correct for the absorbance interference caused by inhibitors, as long as it is not too large.