Dear Lalrinzuali Khawlhring,

Please read the following text:

Is there any formula of transforming relative enzyme activity into absolute enzyme activity? Or does it need a SOD standard to get the absolute activity of SOD?

Yes, you would need to use a standard to find out the absolute activity. Here is the recombinant protein you can use as the standard: http://www.biovision.com/superoxide-dismutase-human-recombinant-3644.html. You will have to do a kinetic experiment to get this absolute activity. I can give you an exapmple of how to calculate the activity:

Plot a standard curve with different conc of the SOD using exactly this assay protocol.

For all samples, do a kinetc assay: Measure OD 450 nm at T1 to read A1, measure again at T2 after incubating the reaction at 25°C for 10 - 20 min (or incubate longer time if the SOD activity is low in sample) to read A2, protect from light. The signal increase is due to SOD, ∆A = A2 – A1

Note: It is essential to read A1 and A2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A1 and A2 in the reaction linear range.

Calculation: Subtract 0 standard readings from the standards. Plot the SOD standard curve. Apply the ∆A to the standard curve to get B nmol of activity generated between T1 and T2 in the reaction wells. SOD calculation:

SOD Activity = [B/(T2-T1)xV] x sample dilution factor = nmol/min/ml = mU/ml.

Where: B is the SOD amount from SOD standard curve (in nmol).

T1 is the time of the first reading (A1) (in min).

T2 is the time of the second reading (A2) (in min).

V is the sample volume added into the reaction well (in ml).

For more information, please use the following link:

http://www.biovision.com/superoxide-dismutase-sod-activity-assay-kit-2852.html?gclid=CjwKEAiAluG1BRDrvsqCtYWk81gSJACZ2BCet4fl6MfQVA5UQt39P3MQrF-JDcKS9B50Mhnrkjuz8xoChJrw_wcB

Hoping this will be helpful,

Rafik

Dear Lalrinzuali Khawlhring

I work on SOD activity by pyrogallol method

The attached photo is the calculation of methed I hope it benefit to you

Dear Lalrinzuali

Check in the reference of  your procedure the substance that inhibit SOD  and the percentage of inhibition. Or send me the reference to advice you  

To calculate SOD activity, after determine the % inhibition Plot the % inhibition v the protein concentration.

Determine the protein concentration that gives rise to 50% (Km) inhibition or use curve fitting software such as Prism or Enzfitter using the Michaelis-Menton equation.

TROUBLESHOOTING

To determine the SOD activity, convert the Km values (currently in μg) to Units

mg-1 protein, Thus, to obtain the activity value for a sample, divide 1000 by the Km

(50% ½ max value) giving the value in U mg-1 protein.

To be sure that the value obtained is accurate, analyze all the samples in a study on the same day and repeat with three independent sample sets.

SOD enzyme calculation

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.

I would be appreciate if let me know the reference related to formula(jpg file)

I would be thankfull if someone kindly elaborate about the reaction mixture. How to prepare?

The reaction mixture (3 mL) contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 0.1 mM EDTA and 0.05 mL of enzyme extract(Supernatant: see above Total protein).

Setareh Farahmand Great. Very happy to have good answer.

How can i represent SOD value in percentage. What is the normal healthy range for SOD in %??

In my case, I expect 20 to 60 % depending on the different treatments that I use for my experiment. I think it depends on the type of study.

Will the absorbance at 560nm will increase and the activity will decrease with increasing stress and time. Do the absorbance and activity are reciprocal. Please reply.

@nasreen. Yes. More activity means lesser colour intensity, hence, lower absorbance

Thanku very much dear @Lalrinzuali Khawlhring

Hi

Superoxide dismutase (SOD) activity assay

Phosphate buffer (0.667 mL, 75 mM, pH 7.00), dH2O (0.06 mg mL-1 of protein), xanthine (0.1 mL, 150 mM), and NBT (0.1 mL, 1.5 mM) was transferred into a quartz cuvette (d = 0.1cm). After equilibration (25°C, 5 min) of the reaction mixture, xanthine (0.023 mL, 0.4 U mL-1) oxidase was added to it and the optical density (ΔOD) was recorded (per second, 3 min, 560 nm) against a blank sample containing dH2O in reaction mixture instead of xanthine oxidase. After 3 mins, the extract (0.05 mL) was added into the reaction mixture and the decrease in optical density (ΔOD) was recorded up to 7 min. The specific SOD activity was calculated as units per mg of protein.

For calculation follow this artcle DOI: 10.1016/j.bcab.2020.101661

Uttam Roy Will you please help me with the formula for the calculation of Ascorbate peroxidase by Nakano and Asada(1981)and Polyphenol oxidase by Saeidian (2013)?

Thank you in advance