Hi Liger,

I found the answer.

This answer is from Dr Neha Gupta

Dear Sir,

NBT method for SOD assay is based on principle that NBT undergo photoreduction (which is a blue coloured formazan) on exposure to light by superoxide radicals. It competes with enzyme SOD for superoxide anions. In presence of SOD in reaction mixture, NBT will produce less amount of coloured complex than control.

% inhibition of NBT reduction by SOD = control OD- treatment OD/ control x 100 =X% inhibition.

50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit.

In your case the SOD calculation will be = (0.389-0.120)/0.389 * 100 =69.15%

50% inhibition =1 Unit of SOD

69.15% inhibition = (1/50)*69.15= 1.38unit

1.38Unit is from =10ul of enzyme extract

Total volume of enzyme extract=1ml=1000ul

(1.38/10 *1000)= 138units of SOD

1ml of extract is from 0.5g of tissue

So SOD units/mg = 138/500= 0.276 units/mg Fresh weight

Hi there,

There are some information missing. To convert OD into moles, you need the specific epsilon of reduced NBT at 560nm, you also need the volume of the the extract used during the assay and as one Unit is generally one µmol per minute there is also the need of the time of the reaction... And make sure the rate of the reaction is constant during the time considered (ie measuring initial velocity of enzymatic reaction).

Dear Liger,

Thank you for your answer. The volume of extract for assay was 0.01mL.

This enzyme measured by End point method not Kinetic.

So if its endpoint what is the unit you intend to express activity with? In my opinion, if you want to express properly enzyme activity it has to be a link with enzyme velocity...

Dear Liger,

I want to report the activity of this enzyme. I need the correct method for transform the absorbanse to Unit/g. fw.

So what is one Unit? For me, one Unit is one micromole/minute but as there is no time notion in your assay, it's going to be difficult to use the same Unit...

One unit of SOD activity would be defined as the amount of enzyme required to cause 50% inhibition of the NBT photoreduction rate.

So there is the notion of rate in your measurements so there must be the time parameter somewhere...

Hi Liger,

I found the answer.

This answer is from Dr Neha Gupta

Dear Sir,

NBT method for SOD assay is based on principle that NBT undergo photoreduction (which is a blue coloured formazan) on exposure to light by superoxide radicals. It competes with enzyme SOD for superoxide anions. In presence of SOD in reaction mixture, NBT will produce less amount of coloured complex than control.

% inhibition of NBT reduction by SOD = control OD- treatment OD/ control x 100 =X% inhibition.

50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit.

In your case the SOD calculation will be = (0.389-0.120)/0.389 * 100 =69.15%

50% inhibition =1 Unit of SOD

69.15% inhibition = (1/50)*69.15= 1.38unit

1.38Unit is from =10ul of enzyme extract

Total volume of enzyme extract=1ml=1000ul

(1.38/10 *1000)= 138units of SOD

1ml of extract is from 0.5g of tissue

So SOD units/mg = 138/500= 0.276 units/mg Fresh weight

thank you for the calculation.. 

you're welcome

The answer from Dr Neha Gupta is very informative, I have similar doubts but now everything is absolutely clear. Thanks again

I also have the same problem. i carried out SOD using NBT only.

with three sets of two groups.

group one in dark and group two in light.

by formula says

%of superoxide radical scavenged= 1-Al-Ad  /  al-ad *100

where as Al= absorbance of sample in light

                  Ad= absorbance of sample in dark

                  al= absorbance of control in light

                  ad= absorbance of control in dark.

do above formula is correct, can anyone suggest formula form it.

can any one suggest formula for it

Hi, EveryoneI

I need some help with my SOD experiment.

I am using the 19160 SOD Assay Kit-WST for my SOD experiment between young flies and old flies. I found that young flies has % inhibition of 60, while old flies has % inhibition of 30. Using the following equation: SOD activity (inhibition rate %) = {[(Ablank 1 - Ablank 3) – (Asample - Ablank 2)]/ (Ablank 1 - Ablank 3)} x 100

Here is my question, is that mean old flies has more SOD because of the low conversion rate of WST-1 to WST-1 formazan. Please help.

For this experiment, I measured the Endpoint, not kinetic. I want to compare the SOD activity between young and old flies. Also, is it possible to report my results as Units/ml Enzyme? If so, what equation should I use?

Thank you so much for your time.

Hi everyone!

I really appreciate the calculation. I am wondering if I use this calculation, do I still need the standard curve to calculate SOD activity? What is the difference between this calculation and using the standard curve? 

Thank you so much.

Hi Mohammadreza Pourghayoumi Sir, can you please give some reference which supports your calculations for SOD activity.

Thanks in advance

why all enzymatic and non-enzymatic antioxidants are represented as per mg of protein? what is the reason for measuring protein?

I am agree with Piotr Rzymski suggestion, your calculation would be inconclusive without protein yield.

Enzymes are proteins. So, we collect protein sample to calculate enzyme conc. or enzymatic activity. By calculating enzyme or enzyme activity per mg protein, we equalize the content of protein of the known sample (which is use to draw a standard curve or to compare) and the test sample to draw a conclusion.

Hı

I have a question. How you know amount of leave sample for extraction?

Hi Sahra, in Sri Lankan Pharmacopoeia, they usually take roughly 250g of the plant and air dry it to produce appr. 60g of dried plant to make an extraction. Hope this helps!

Hi Jivendra, thank you for your feedback. I thought for cell culture. So I was surprised :D