SOD assay problem

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fatma_elzahraa
fatma_elzahraa's picture
SOD assay problem

Dear All,

I tried to measure the activity of superoxide dismutase (SOD) in bone homogenate (10%)....
I found a good paper that took Marklund method as reference with some modification

Basic & Clinical Pharmacology & Toxicology, 103, 55-60 (2008)

α- Tocopherol
Sandra Maniam, Norazlina Mohamed, Ahmad Nazrun Shuid and Ima Nirwana Soelaiman

Palm Tocotrienol Exerted Better Antioxidant Activities in Bone than

,

the paper said: 2 ml 0.01MTris-HCl buffer pH 8.5 + 750 ul sample + 750 ul water + 500 ul pyrogallol (2mmol in 0.01M HCl)

and read the absorbance at 420 nm at 1,2,3 min then calculate dA/dt, % inhibition, SOD concentration then its specific activity.

My problem is the difference between readings at intervals (1,2,3 min) for the sample  were larger than those for the blank

as I calculate the % inhibition from the equation:
% inhibition= 100 - (dA/dt for sample / dA/dt for blank * 100)  

so the % inhibition gave a negative result (unreasonable one)

i changed alot of factors including the sample volume and pyrogallol concentration.... but all trials were in vain...

please, is there anyone can help to solve this problem.... 

Sami Tuomivaara
Sami Tuomivaara's picture
fatma_elzahraa,

fatma_elzahraa,
Could you please clarify what is your blank.

If you look at your equation carefully, it gives negative value no matter what values you plug in (as long as the dA/dt_sample is higher than dA/dt_blank). Maybe the problem is in the equation rather than experimental setup.
Cheers,

fatma_elzahraa
fatma_elzahraa's picture
dear,

dear,

first thanx alot for ur care...

then, my blank contains buffer and water or homogenizing buffer and pyrogallol without sample....
it should give dA/at higher than that of sample... but i got the opposite...
i increased the sample volume.... and i was working with 20 mmol pyrogallol then replace it with 2 mmol but the results still strange....
i don't think about the sample needs dilution as the absorbance difference becomes so small or undetectable in samples with high sod content.... but my samples read large differences in absorbance...

do u recommend reducing pyrogallol conentration to 0.2 mmol may solve the problem or not....
really iam so confused and need any help in near time....

best regards

Sami Tuomivaara
Sami Tuomivaara's picture
fatma_elzahraa,

fatma_elzahraa,

First of all, I didn't put my self out as clearly as I wished in my earlier post. What I meant is that I think if you are calculating per cent inhibition, the formula should be something like this

%-inhibition = (1 - (dA/dt_blank - dA/dt_sample) / dA/dt_blank) * 100

Also, on the second thought, you're right, there seems to be something weird going on in the experiment as well, it can be lot of things. Can't help you there right now, I'll put my thinking hat on. I'm sure there are other people reading this who have an opinion.

Cheers,

sadia khan
sadia khan's picture
SINCE YOU ARE MEASURING THE

SINCE YOU ARE MEASURING THE INHIBITION ,SO THE BLANK WILL SHOW HIGHER READING IN CONTRAST TO THE SAMPLE.
I WOULD LIKE TO KNOW HOW TO CALCULATE THE SPECFIC ENZYME ACTIVITY OF SOD USING MARKLUND METTHOD

fatma_elzahraa
fatma_elzahraa's picture
Dear,...

Dear,...
Superoxide dismutase was so easy to be dtermined by Murkland....
u should prepare ur buffer (Tris-HCl 0.05 M containing 1 mM DTPA pH 8.5, this can be prepared by calculating the wt of Tris and DTPA to have their concentration but don't complete the volume to the end i.e. leave a small water amount before adjusting the total volume... now adjust pH to 8.5 by HCl then add the small water amount remained to reach the total volume)
pyrogallol prepared 20 mM in 10 mM HCl
depending on ur tissue SOD level the volume of buffer, sample and pyrogallol added to the reaction mixture can be determined... for example, u can use 1 ml buffer and 10 or 20 ul of ur homogenate then add 10 ul of pyrogallol... at the same time u put pyrogallol and mix it well with the reaction mixture, adjust ur stopwatch and measure the absorbance at 420 nm at timed intervals (may be 1, 2 and 3 min readings or 1.5, 2.5 and 3.5 min readings... u can take both and calculate the activity which may give u the best results)
for calculating the activity after that... u should read blank first (buffer + water or ur homogenizing buffer + pyrogallol) that should read higher than ur samples...
then calculate dA/dt = ((reading at last interval - reading at second interval)+(reading at second interval - reading at first interval))/2 min.... the duration u take readings in.....

then calculate the inhibition percent = (dA/dt of blank - dA/dt of sample) / dA/dt of blank * 100

u should do the same for a series of sod standard solutions prepared in deionized water.... and test ur concentrations to know the concentration range u can take it for measuring....
if u find that dA/dt of samples are so small or nearly zero, u should dilute ur samples...
and if u find that dA/dt of ur samples are higher than that of blank, so u should increase ur sample volume or contration in the reaction mixture.... ur accepted range of inhibtion percent should be between 20% - 60%..... if it is higher, dilute ur samples...
if smaller, increase ur sample volume...

then construct a calibration curve, and get the sod concentration at which 50% inhibtion is achieved...
this concentration can be considered as 1 U
from this u can get the activity in units.... then divide it by the protein concentration in ur homogenate, so u can get the specific activity....

i hope i could give u a detailed summary of the procedure....

u just need to standardize ur assay depending on ur tissue level of sod....

N.B. if u calculate the dilution factor of pyrogallol in the reaction mixture u will find its final concentration is 0.2 mM
don't hesitate to ask about anything
best regards

sandeepgoyalpgi
sandeepgoyalpgi's picture
 Dear Dr Fatma,

 Dear Dr Fatma,

I am a graduate student from India. I am busy in doing SOD assay. My dA/dT readings of sample are quite higher than control ones. Do we always need to use standard SOD. Can we use any formula based method too?

Thnx.

Sandeep 

fatma_elzahraa
fatma_elzahraa's picture
dear sandeepgoyalpgi

dear sandeepgoyalpgi

look, u should increase ur sample volume in the reaction mixture...
can u tell me about the tissue u measured sod in.... i may help...
i got the volumes for my assay....
u can try them out...
800 ul of 50 mM tris-HCl buffer containing 1 mM DTPA (pH 8.5) + 600 ul sample + 14 ul pyrogallol (20 mM) in 0.01 M HCl and adjust ur stop watch and read the absorbane at 420 nm at 1, 2, 3 min after mixing or at 1.5, 2.5, and 3.5 min after mixing....
and try varying the volumes but not change the pyrogallol concentration ....
i.e. the total volume in my assay is 1400 ul so pyrogallol volume should be added is 14 ul (1/100 of the total volume)
i hope u get better results ....
and u should construct ur standard curve.... no formula can give u the concentration in units....
best regards

sandeepgoyalpgi
sandeepgoyalpgi's picture
 Dear Dr Fatima,

 Dear Dr Fatima,
Thanks for your suggestions.
The enzyme source is rat brain. This time I used (120 ul enzyme source + 80 ul Buffer = total 200 ul in 200ul of cuvette) and I used 20 ul of pyragallol. I dont find dA/dt of blank (without enzyme source) more than sample ones. But I observed that in blank, change in aobservation does not occur immediately but after 3 minutes, there was significant change. If I compare this with sample, I found % inhibition was nearly 70% in all samples.

Thanks 

Sandeep

brindaram
brindaram's picture
I am a beginniner for SOD

I am a beginniner for SOD calorimetric assay.. I have prepared pyrogallol (10mg) in 100 ml of 0.05M Tris-HCl. The problem is the color starts changing from 5th minute despite wrapping up with aluminium foil. I m in urgent need to do SOD assay asap. Please help me with this. 

Radzik
Radzik's picture
This buffer is not suitable

This buffer is not suitable for pyrogallol solution. Dissolve pyrogallol in 10mM HCl this should help - in acid pH autoxidation of pyrogallol is inhibited.

brindaram
brindaram's picture
Dear Radzik, thank u for the

Dear Radzik, thank u for the reply! but if 10mM Hcl is used the final concentration changes altogether. Can u please provide with elaborate data? wat should be the conetration of pyrogallol in 10mM Hcl? how mcuh ml should be added? I wonder how no one discussed on trouble shooting this probelm while using pyrogallol? I found the readings to change once every 15minutes. Autooxidation was so prominent. I changed the pyrogallol every 20 minites and for each 20 minutes i took 2 pyrogallol auto oxidation readings which vary at 1st and 10th or 20 th minute. I am planning to average the pyrogallol readings. Please suggest me with how to do SOD assay!
Also i am facing small trouble in catalase assay? some have not mentioned about heating of final solution after adding dichromate. Is it an essential step? Please let me know the formula for catalase calculation. Is nt there any video links to do SOD and CAT by popular methods and their respective trouble shooting!? I would highly appreciate if one can provide with such kind of link! Please share the knowledge guys! I am struggling a lot to handle 28 samples at a time. I am very new to doing this and i dont have proper facilities to perform these experiments. 
Please let me know if there are any methods to do assay using elisa plates with minimum quantity of sample volume. Scientific papers i got are little confusing.

Radzik
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Dear Brindaram

Pyrogallol solution in 10 Mm HCl should be 24 mM, (303 mg/100ml). The most important is appropriate aeration of assay buffer (50 mM pH 8,2 with 1 mM EDTA), by vigorously shaking (10 – 15 minutes). I’m adding too to, the assay buffer 10 micro molar in solution bovine catalase (2.5 mg protein/ per ml). I adapted classic protocol to microplates. Start - move 6 ul of sample to assay cell, then 289 ul of assay buffer (with 33 ul catalase solution per ml in assay buffer), Then add 9 ul of pyrogallol solution. For absorbance reading I use Infinite® M200 PRO spectrophotometer with wave length 420 nm, temp. 25oC, kinetic program conditions, with absorbance readings every 10 seconds by 3 minutes. This protocol works very well.
Catalase activity I assay by measuring decreasing of absorbance at 240 nm which is a result of the decomposition H202 (J Biomol Tech. 2007 September; 18(4): 185–187). Shortly – prepare 50 mM phosphate buffer pH 7.0 and dissolve H2O2 to final concentration 5mM. Next pipette 5μl sample or less, add 250 μl of the buffer with H202 mix well and immediately read absorbance by 2 minutes with 15s lag phase. In the same time should be prepared blank with buffer volume against sample. Remember about properly sample dilution, means that decreasing of absorbance should be linear. In one time I assay no more than 7 samples and blank. For both methods I use multichannel pipettes. Formula for activity calculation  -
 
Units/ml = (dA/min(blank) – dA/min(sample)) *sample dilution *assay volume
                   ______________________________________________________
                        sample volume *molar extinction coefficient 0.0436 mM (for path length 1 cm)
 
One unit of catalase decompose 1.0 micromole of hydrogen peroxide to oxygen and
water per minute at pH 7.0 at 25 °C at a substrate concentration of 5mM hydrogen peroxide
 
You must count by self molar extinction coefficient for cell in microtiter plate by simply counting column height of liquid (in cm) and then multiply it by the value molar extinction coefficient. This correction of molar extinction coefficient is necessary because absorbance depends on path length.
In the protocol with dichromate is necessary boiling the sample after addition dichromate. This method works well, but is more complicated – more steps and chemicals. If You have inYour laboratory  microtiter plate reader with possibility reading absorbance at 240 nm try this metod. You need for this  UV transparent microtiter plate. I use plates from Greiner.
 
I hope that this will helpful for You

brindaram
brindaram's picture
Dear Radzik,

Dear Radzik,
That was really helpful. But i have a couple of doubts.. u have mentioned "Start - move 6 ul of sample to assay cell, then 289 ul of assay buffer (with 33 ul catalase solution per ml in assay buffer)"  My sample is rat liver extract in which i ve to estimate the SOD amount. Wat is this assay buffer?? Y should we add sample as well as catalase to assay buffer? I have little problem in understanding that portion. Please could u explain me further? 

Catalase experiment sounds easy and i ll reduce my sample size this time. That was a lot helpful information. Thank u so very much! 

Radzik
Radzik's picture
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Dear Brindaram
A base of SOD assay buffer is 50 mM pH 8,2 with 1 mM EDTA and  before You will start with SOD assay add to 10 ml of this buffer 33 μl of mentioned catalase solution and mix well. Then move 6 ul sample to microtiter plate add appropriate assay buffer volume and for the end  volume of pyrogallol solution.  In original protocol catalase solution was added after addition of assay buffer to sample, but this is additional step that can be easily omitted.
Liver is very rich in catalase activity, I work with this material from several species (fishes, mammalians, birds) and I think that You must dilute sample at least 1:40 for catalase assay. In case when decreasing in absorbance will  still not linear don’t dilute more, only change sample volume. On the beginning try different dilution with only one sample and when You catch correct dilution fix it to all samples.

Radzik
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Dear Brindaram

Dear Brindaram

The assay buffer is of course  50 mM TRIS - Cl pH 8,2 with 1 mM EDTA

brindaram
brindaram's picture
 Thank you Radzik! I have one

 Thank you Radzik! I have one common qestion. I have stored my liver samples in -80 degree C for storage. One of the researcher i met this week told me that it is not a fair idea to store livers. He added that sure the readings will not be the same and reliable if we do assay with stored liver rather than doing it fresh on the day of sacrifice. I was not able to accept this fact because we store plant samples for several days for the purpose of antioxidant assays. How about animal samples. Do u have any suggestions? Also i have sotred a portion of livers in formalin for histo patho analysiss. Am I doing things in right way?

Radzik
Radzik's picture
Dear Brindaram

Dear Brindaram
I store my samples at -80oC and I don’t have problem with stability of the samples for enzymatic assays at last for 1 month, but when I have enough time I homogenize tissue sample immediately and store homogenate until day of enzyme assay. I avoid refreezing the samples because this can destabilize them and I prepare a few tubes with small amount homogenate for each sample. Maybe the researcher was thinking about stability of the lipids peroxidation level (LP) that can be expressed as MDA concentration, (oxidative stress marker). LP level is not stable and should be measured on fresh tissue. Freezing the sample at -20 can promote elevation of lipid peroxidation but at - 80 the sample should be stable. You can use BHT  to prevent lipid peroxidation in tissue homogenates. I assume that You study influence some factor on oxidative status of liver .
 

brindaram
brindaram's picture
Oh that was a real good

Oh that was a real good information Radzik. Many were confusing me regarding storage conditions. And now i think i must skip doing LPO (TBARS) assay any further cuz my samples are already 25days old and i ve have stored them in ziplocks without any solution. I adopted this method from a lab in near by university. 
Well yeah the professor insisted me that there will be variation in enzyme levels. I hope i will have atleast the relative values unchanged.
I wish i had found this website much earlier to get solutions in regard to my experimental plan. 

Thank u...

nkb
nkb's picture
 Hello..

 Hello..
myself Nidhi..
I am working on SOD1 protein.
I carried out pyrogallol assay to find out the specific activity.
I m doing this experiment for the first time and I followed the Marklund's method.
I m facing the problem in calculations.
I hv been able to find out the protein concn where 50%inhibtion take place from change in absorbance values but I unable to correlate this value with specific activity.
Though this value corresponds to one unit.
But How to get specific activity from this value.
Actualy I am not core biologist.
Can you please tel me how to proceed  with the calculations.
And  one more  query,in the marklund's paper which I followed, they have mentioned that its better to adjust the pH of Tris buffer with cacodylic acid as sensitivity decreases with HCl acid.Is that so because I have used HCl acid.

indu
indu's picture
 how do I calculate SOD

 how do I calculate SOD activty.
I've taken 25micro L of homogenate ..... added 2ml buffer......addded 750micro L of water and finally added .5ml of pyrogallol (totaly 3.5ml of volume). I did the kinetic studies for 120mins at 30 sec intervals. I got .025 for blank pyrogallol (no sample) and with sample I got .010. That comes to about 60% inhibition. Anyone tell me how to calculate the SOD activity from this info.