Answering 9 RG questions related to spin-trap DMPO

Alexa knows... It knows more than you, more than your mother, more than Easter-European grandma. A bit less than CIA or KGB, but still, it knows a lot.




Last time I checked, there were 1.070 links to Steemit. I added 4 new link, and Alexa counted 1.074. Let's make 9 more.




For those of you who have no clue what I'm talking about, https://steemit.com/ is a blogging platform that pays in cryptocurrency called Steem. https://www.steemstem.io is STEM community on Steemit - clear as sunny day.




Now it's time for Questions!



> What is the procedure to prepare water samples and spin traping DMPO for OH using in electron spin resonance (ESR) measurement? (link)


> Should I add DMPO before adding oxidant or before analysis when using ESR spin trap method to monitor the radical species in reaction? (link)


> What should be the concentration DMPO during ESR spin trap? And what is the protocol for ESR Study? (link)


> How can I prepare cells suspension for EPR analysis? (link)


> How can I reoxidize DMPO inside cells? (link)


> Anyone has experience on EPR on total tissue to measure superoxides and hydroxyl free radicals (link)


> I am performing DMPO spin trap EPR experiments where the hypothesis is to detect superoxide adducts, but instead, why am I detecting sulfite? (link)


>  How to analyze EPR spectra and calculate the hyperfine splitting constants? (link)


> How can I identify contribution of different radical components to EPR spectra? (link)

Now it's time to answer...

The style will be similar to my previous post about the questions related to PCA. I will answer all at once, in a relatively meaningful text.




Ok, you can mix spin-trap, DMPO, DEPMPO... with water. Now, if you want to generate *OH radicals, all you need is Fenton reaction. If you want to see the recipe, follow the link. If you want to generate similar radicals, just change the solvent. Use methanol/ ethanol instead of water (read the paper from the link).




EPR is not detecting short-lived free radicals. This is why you are performing spin-trapping - to covert short-living radicals into long-living radicals. In the perfect world, spin-trap will have only one adduct per radical. It will be stable (not converting spontaneously into another adduct) and it will allow splitting of the signal. This is why you should add the trap before the reaction starts. 




Concentration should be as low as possible in order not to interrupt the reactions. In practice, you sample for X-band EPR will be about 50 μl, and you can add somewhere between 0.5 and 2 μl of the trap (usually 1). My personal record for the highest ratio between the trap and the substance was 20% for CLASSIFIED.




Concerning cells, there should be no problems, in general. Nothing special is required... Do you have a proper concentration of cells (centrifuge helps)? Have you accumulated the signal long enough? For how long your cells were in "wrong" conditions (temperature, gasses...). Have you scraped them? Is the medium unusual? 




Superoxide and Hydroxyl with DMPO... Don't fall into a trap and use the wrong trap! You can't do this properly, those two adducts interconvert. Use DEPMPO instead.


And finally, after all a lot of suffering - you need to analyse the spectra.
In biological systems, there will be two: DMPO-OH and DMPO-OOH.
In biochemical systems, count on *C, *OC...
My measurements on CLASSIFIED  confirmed at least 6 classes of radicals - at once. God help me... Here is the real workflow, forget the books:

1. Go to https://tools.niehs.nih.gov//stdb/index.cfm and write down the parameters for all the radicals you can expect to find
2. take the solvent into account, if it's not water - good luck. Gaussian is not going to help you. However… All the parameters will follow the order…
3. Calculate how wide is the widest
4. And calculate what you can expect at the centre
5. Now… Start from the widest peak
6. Take a look at the zone of the 2^nd/3^rd peak occurrence
7. After you have finished that, if you still have radicals - go to the center

Why I needed to work in this way? It's a simple quesion. Once there is 4+ signals, math can't help you anymore and you can fit everything with anything.In that case, pen, pencil, highligher and ruler are the best solution.