I would advise you to read "Assay of superoxide dismutase activity in a plate assay using WST-1" Free Radic Biol Med. 2017 Feb;103:188-191.

Thanks Alexander!

I read the paper, and it offers a lot of suggestion, but I do not think (unless I missed it) it may help explain our background issue. We believe (and should have thought about it earlier) that having mitochondria isolate in the sample generates superoxide (CI and CIII are certainly generating ROS and superoxide) endogenously, and there is the likely possibility that we have other enzymes, including endogenous xanthine oxidase, which may generate more superoxide.

We are planning to boil a couple of samples to see if we can inactivate endogenous generation of superoxide. We also want to try and inhibit CI (Rot) and CIII (Ama) to see if we stop generation of superoxide by the ETS. Unfortunately, we think that in the first case we may inactivate all sort of things including MnSOD, and in the second case we may not eliminate all possible endogenous sources of superoxide.

If you think of any other possible solutions I am more than happy to listen to your suggestions!

thanks a lot

cheers

c

Hi

I had this problem previously. This assay is not specific as tetrazolium salts are non-selectively reduced by many other factors. I did this test before step by step as this article said "Assay of superoxide dismutase activity in a plate assay using WST-1" Free Radic Biol Med. 2017 Feb;103:188-191".Only the standards were ok because no interference is existed in them.I tried other tetrazolium salts such as MTS and MTT. MTS was acceptable in lysed RBS as positive control, however, it did not produced acceptable results in liver and serum samples again. I also have used abcam kit that has used the same principles as cayman does. Same as always. Finally, it draws me to investigate another dye that is completely specific for this enzyme that for now I can not reveal because of patent process. I recommend to test cytochrome C method that in my opinion is much more sensitive for that. It must be noted that NADPH/NADH oxidase are used WST-1 as substrate so this agent can also be reduced by many enzymes.

Best

Interesting

Thanks Farjam,

It certainly doesn't look as easy as the Cayman people are making it to look.

I still think we have a problem upstream of what you mention with the specificity of the tetrazolium salts...and that is that we generate superoxide endogenously.

thanks

c

Hi Cesare,

What did you do with your mitochondrial preps before you use them for SOD activity assay?

Alexander,

i don't quite understand the question.

I isolated mitos with the following protocol:

Buffers and Reagents:

• Solution B

- 20 mM Hepes-KOH pH 7.6

- 220 mM Mannitol

- 70 mM sucrose

- 1 mM EDTA

Check pH is 7.6, adjust with KOH if needed

Add PMSF to a final concentration of 0.5 mM.

• Sucrose Buffer:

- 10 mM Hepes pH 7.6 (with KOH)

- 0.5 M Sucrose

Mitochondrial isolation method:

Perform steps at 4°C or on ice

1) Resuspend ~40 mg of kidney cortex in 3 ml Solution B

2) Transfer to glass homogenizer. Homogenise with 30 strokes using a drill fitted Teflon pestle on ice (use quite a fast speed but make sure it does not become hot and that there are no pieces of muscle left and it is well homogenised)

3) Transfer homogenate to 2x 1.5 mL Eppendorf tubes and centrifuge at 1000 g for 10 min at 4°C

4) Collect supernatant (containing mitochondria) and aliquot into 2x 1.5 ml tubes. (Do not disturb the pellet at bottom).

5) Centrifuge at 12,000 g for 15 min at 4 °C

6) Remove supernatant (absolutely do not disturb the pellet, as you would loose a lot of mitochondria) and resuspend pellet (crude mitochondria) in 100 uL of sucrose buffer combining to a single 1.5 ml Eppendorf tube for a total of 200 µl

7) Perform a BCA on 5 µl

At this stage we proceed with the Cayman kit as per instruction (10 uL). Although it think going forward I want to make the conc of all my samples the same (say 2 ug/ul) and continue from there, as the assay seems to have a very poor linearity range.

have I answered your question?

c

Yes, you did answer. Apparently the reduction of tetrazolium salt you observed was due to direct superoxide-independent reduction by membrane redox chain. You have to destroy mitochondria and remove all membranes before doing SOD assay. Reading papers in depths always a good idea before jumping to conclusions - this is for Farjam.

Thank you alexander for the tip

But I did that before and the result was the same. As the serum samples (no mitochondria) are always produce the same consequences. Another point must be noted, the reaction is started when xanthine oxidase exposed to its substrate (xanthine) that is an independent reaction. By the way tetrazolium interferes non- specifically by the other factor rather than mitochondria.

Let me to clear the situation: To clear any disruption by your sample you can run sample well in parallel with your sample. This well contains all components except xanthine oxidase. This well also does not produce any color that shows no reaction is started. But the problem is xanthine oxidase. When it is added to the well reaction is developed by the way (samples contained SOD inhibit color development). This enzyme in the presence of xanthine will produce superoxide, H2O2 and urea. To exclude products interference, I also added these products separately to each well at high concentration but no color development was occurred. The only reason that I could imagine is non-specific secondary reaction is occurred after the addition of xanthine oxidase that is unknown to me. This non-specific reaction is just coupled with tetrazolium not with another dye or method.

Hi Farjam,

I'm not quite following your description of the problem. But based on the info you supplied the xanthine dehydrogenase activity could be involved. The enzyme, apart from superoxide generation, also catalyses the reaction xanthine+H2O+NAD+→uric acid+NADH+H+

If some membranes are present in your sample then superoxide-independent tetrazolium salt reduction would occur.

It is really great that you are very careful with running controls.

Hi Alexander

If the mentioned reaction is the case, adding xanthine to the well that contains everything except the xanthine oxidase must develop the color production. But it did not! It is so confusing for me and took my time for four months and at last I used another dye and everything gone right.

Hi Farjam,

The same protein may act as xanthine oxidase or xanthine dehydrogenase. If no tissue sample present you could see tetrazolium salt reduction which is fully inhibited by SOD. With the tissue sample containing NAD+ and membrane vesicles one may observe SOD- independent tetrazolium reduction.

Don't be frustrated. You are doing a good job by trying to solve the problems rather then overlooking them,

Thanks Alexander (and Farjam) for helping out, and sorry for the delayed response, but I have been sick for the last 4 days.

Alexander,

you mention in your last reply to me that "You have to destroy mitochondria and remove all membranes before doing SOD assay".

As I was mentioning in a previous answer to you (specifically my second answer of this thread) we are planning to boil the sample to inactivate the mito (and also to add CI and CIII inhibitors). However, as I was saying my fear is that if I inactivate the mito I may inactivate the SOD enzyme too.

You seem to suggest to destroy mitos and remove all membranes.

Would 5-7 min at 95-100 ^C achieve that? I am sure we would destroy the ETS and the mito, but how do I remove the membranes? further centrifugation step after boiling?

If you have a protocol to destroy the mitos and remove the membranes, I would be very grateful if you could share it w/ me, as I am not quite sure how to achieve this.

Would this be selective, in that the SOD enzyme would still be functioning?

Thanks a lot for your help with this!

cheers

c

By saying "destroy" I mean to break down mitochondrial integrity rather that inactivate them. You could sonicate mitochondria and then pellet membranes for 1h at 100,000g and measure SOD activity in the supernatant. Make sure that the protein concentration in your samples is high enough as mitochondrial SOD activity significantly lower compare with cytosol SOD.

ok

we will try to do that...I am not sure we can find an ultracentrifuge...but I will try.

thanks for your help so far!

c

I think you may try size exclusion as a replacement for ultracentrifugation.