According to the literature, superoxide dismutase exists in all oxygen-metabolizing cells to protect cells from excessive superoxide. Under the action of SOD, two superoxide anions were converted to oxygen and hydrogen peroxide.

In mammals, there are three different forms of SOD: CuZn-SOD, Mn-SOD and EC-SOD (an extracellular form of SOD). Cu-Zn SOD exists in the cytoplasmic and mitochondrial membrane spaces of the cells, while Mn-SOD is located in the mitochondrial matrix.

1.   Determine the optimum dilution multiple of the sample before formal experiment. Calculate the inhibition ratio of serial dilution multiple of sample, and choose the optimum dilution multiple when inhibition ratio in the range of 30%~40%.  

2.   EDTA should not be as anticoagulation, suggest to use heparin plasma.

3.   The prepared enzyme working solution must be use out within 20 min.

                                                                           [Note]: A, Controlblank wells; B, Control wells; S1-S92, sample wells.

1.   Controlblank well: add 5 μL of PBS (0.01 M, pH 7.4) to the Controlblank wells.

Control well: add 5 μL of PBS (0.01 M, pH 7.4) to the Control wells.

Sample well: add 5 μL of Sample to the Sample wells.  

2.   Add 90 μL of Reagent 1 working solution into each well of Step 1.

3.   Controlblank well: add 30 μL of Non-Enzyme working solution.

Control well: add 30 μL of Enzyme working solution.

Sample well: add 30 μL of Enzyme working solution.

4.   Shake for 10 s with microplate reader and cover the plate with sealer, incubate for 50 min at 37 ℃.

5.   Add 180 μL of Chromogenic agent into each well of Step 4.

6.   Shake for 10 s with microplate reader and stand for 10 min at room temperature. Measure the OD value of each well at 550 nm with microplate reader.

[Note]: Control well and Controlblank well can be done with 2 wells respectively.

For rat liver tissue, dilute the 10% rat liver tissue homogenate with PBS (0.01 M, pH 7.4) for 160 times, take 5 μL diluted sample, then carry the assay according to the operation table.

The results are as follows:

The average OD value of the sample well is 0.248, the average OD value of the control well is 0.333, the average OD value of the Controlblank well is 0.132, the concentration of protein in 10% rat liver tissue homogenate is 11.61 mgprot/mL and the calculation result is:

T-SOD activity (U/mgprot)=[(0.333 - 0.248)÷(0.333 - 0.132)]÷ 50% × 0.305÷0.005 × 160 ÷ 11.61 = 711 U/mgprot

Detect human serum (dilute for 2 times, V2=5 μL), human urine (dilute for 3 times, V2=5 μL), 10% rat liver tissue homogenate (the concentration of protein is 11.61 mgprot/mL, dilute for 160 times, V2=5 μL) and HepG2 cells (the concentration of protein is 6.09 mg/mL, dilute for 20 times, V2=5 μL) according to the protocol, the result is as follows: