My goal is to characterize a dehydrogenase, we are unsure of which cofactor it can uses but we tried DCPIP as artificial redox cofactor and there is no activity.
I would like to try phenazine methosulfate (PMS) and MTT, which I found associated with glycerophosphate dehydrogenase and methanol dehydrogenase assay.
As I understand so far, potassium cyanide (KCN) is used in redox enzymatic assays to prevent oxygen mediated oxidation of the electron acceptor when this is of a quinone or cythocrome nature (please correct me if I'm wrong). Seems also to be very toxic, can it just be replaced by something else like DTT or manipulated in an anaerobic tent?
Also cyanide gas release from KCN can be prevented by using it in alkaline conditions but I cannot find how alkaline, is pH 8 enough?
Thanks!
I'm not sure if this is relevant to your situation, but I have used XTT + diaphorase to detect production of NAD(P)H from NAD(P)+ by a dehydrogenase.
Hi Luis,
Cyanide binds to complex IV of the respiratory chain and therefore indeed prevents (most) quinone and cytochrome related substrate oxidation. You basically "arrest" the entire respiratory chain, however quinones could still be regenerated by other substrates. Using cyanide should still suppress this activity in most cases though so it certainly won't hurt.
You're right, potassium cyanide is pretty damn toxic. However you only need to be careful when weighing it for stock solutions. I always prepare a stock in 1 mM NaOH or KOH, that is alkaline enough to keep the cyanide in its ionic form and with a concentrated stock you don't alter the pH of your assay when adding a little bit of cyanide. In my experience a final concentration of about 2 - 20 µM cyanide in your assay will suppress pretty much all cytochrome c oxidase activity.
Regarding cyanide toxicity you always need to keep in mind which amounts are present in your stock solutions. If your stock tube contains 5 mg of cyanide then you don't need to worry about that, for example even drinking the entire tube would probably not cause serious harm (not that you should ever do that). Cyanide is "only" an acute poison, meaning there is no harm from repeated low-dose exposure.
Here is the protocol from a kit that uses XTT to measure NADH:
http://www.abcam.com/ps/products/232/ab232856/documents/ab232856%20XTT%20Assay%20Kit-v1%20(website).pdf
It doesn't say what the electron transfer agent is in the protocol.
Information about diaphorase as the electron transfer reagent can be found here, but using a different substrate:
http://www.worthington-biochem.com/DIL/assay.html
I have used the method myself, but I don't think I ever published anything about it.
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