Determination of SOD activities in rat erythrocytes

In order to compare the cytochrome C assay and WST-based SOD assay, superoxide dismutase (SOD) extracted from rat erythrocytes was used. The WST-based SOD assay was performed using Dojindo's SOD Assay Kit-WST. The SOD extraction method from rat erythrocytes and the protocols of the WST assay and Cytochrome C assay are shown below.

Extraction Procedure of SOD from red blood cells

0.9% NaCl and distilled water were kept at 4 oC.




Cytochrome C Assay Protocol

300 mM potassium phosphate buffer, 0.6 mM EDTA, pH 7.8, 0.5 ml

1. add 0.5 ml of 60 uM cytochrome C solution
2. add 0.5 ml of 0.3 mM hypoxanthine solution
3. add 1.0 ml water
4. add the sample solution or standard SOD solution
5. incubate at 25 ^oC for 5 min.
6. add 0.2 ml xanthine oxidase (control the concentration of xanthine oxidase to give the O.D. at 0.025 dA/min)
7. Measure the O.D. at 550 nm for 15 min., and determine dA/min. Use only the linear part of the slope.


WST Assay Protocol

96-well microplate

1. add 20 ul of the sample solution or water (for blank and control).
2. add 200 ul of WST working solution.
3. add 20 ul of dilution buffer to each blank well. Add 20 ul of enzyme working solution to each sample well and control well.
4. incubate at 26 ^oC for 20 min. on a rocking platform.
5. Read the O.D. at 440 nm using microplate reader.


Since the unit (U/ml) of SOD activity depends on the calculation method, it is necessary to use a same calculation method to compare SOD activities. There are two methods to determine the SOD activity. One is where the amount of SOD in a sample solution establishes 50% inhibition of cytochrome C reaction or WST reaction with superoxide. This 50% inhibition point is defined as 1 U. The other is where the unit of SOD activity of a sample is determined by a standard SOD solution. In this experiment, we compared SOD activities determined by the WST assay and cytochrome C assay using the two different calculation methods mentioned above.


Method 1: method where 1 U is defined as the SOD amount that establishes 50% inhibition of the reaction

The dilution rate of a sample solution that establishes 50% inhibition is used for calculation to determine the SOD unit in an assay solution as 1 unit. Assay volumes of Cytochrome C assay and WST assay were 3 ml and 240 ul, respectively. By using a dilution rate during extraction process from rat blood, SOD units in 1 ml of rat blood measured by the cytochrome C assay and WST assay were calculated and compared. The calculation methods are indicated in "Calculation Method for SOD activities in Rat Blood: Method 1."

Method 2: method where the SOD activity is measured using standard SOD solutions

The SOD concentration (U/ml) that establishes IC₅₀ (50% inhibition of the reaction) was determined using a standard SOD (1927 U/mg protein). Then, the dilution rate of rat erythrocyte extract that established IC₅₀ was determined, and the unit (U/ml) of the extract was calculated by the SOD concentration that established IC₅₀ determined using standard SOD. By using the dilution rate during the rat blood extraction process, SOD units in 1 ml of rat blood measured by the cytochrome C assay and WST assay were calculated and compared. The calculation methods are indicated in "Calculation Method for SOD activities in Rat Blood: Method 2."


Calculation Method for SOD activities in Rat Blood

Method 1:

Example using the cytochrome C assay:
The dilution rate of a sample that establishes 50% inhibition of cytochrome C reaction is 2.33 (2.33 times dilution). The dilution rate of a sample solution in the assay was 10 (10 times dilution: assay volume 3 ml/ sample volume 0.3 ml). Therefore, 1 U is defined as the amount of SOD in the 23.3 times diluted sample solution.

Calculation of the unit per 1 ml rat blood:
Since 1 unit of SOD is contained in 0.3 ml sample solution used for the assay, the units per 1 ml is 23.3/0.3=77.7 (U/ml). Therefore, the SOD activity in 1 ml of rat blood can be calculated by using the dilution rate during the extraction process as shown below.
(15/0.5) x (11.5/10) x 77.7 = 2880 U/ml

Example of the calculation using the WST Assay:
The dilution rate of a sample solution that establishes 50% inhibition of the reaction is 10.75 (10.75 times dilution). The dilution rate of the sample solution in the assay was 12 (12 times dilution: assay volume 240 ul/ sample volume 20 ul). Therefore, 1 U is defined as the amount of SOD in the 129 times diluted sample solution.

Calculation of the units per 1 ml Rat Blood:
Since 1 unit of SOD is contained in 20 ul of the sample solution used for the assay, the unit per 1 ml is 129/0.02 = 6450 U/ml. Therefore, the SOD activity in 1 ml of rat blood can be calculated by using the dilution rate during the extraction process.
(15/0.5) x (11.5/10) x 6450 = 222526 U/ml


Method 2:

Calculation example using the cytochrome C method

The amount of the standard SOD that establishes 50% inhibition is 1.73 ug/ml. The units are determined by using the standard SOD (1927 U/mg protein).
1927 x 1.73 / 1000 = 3.33 U/ml
Since the dilution rate of the sample solution that establishes 50% inhibition is 2.33 (2.33 times dilution), the unit of SOD in the sample solution is 3.33 x 2.33 = 7.76 U/ml.
Therefore, the SOD activity in 1 ml of rat blood can be calculated by using the dilution rate during the extraction process.
(15 / 0.5) x (11.5 / 10) x 7.76 = 268 U/ml

Example using the WST assay:
The amount of the standard SOD that establishes 50% inhibition is 0.33 ug/ml. The units are determined by using the standard SOD (1927 U/mg protein).
1927 x 0.33 / 1000 = 0.636 U/ml
Since the dilution rate of the sample solution that establishes 50% inhibition is 10.75 (10.75 times dilution), the unit of SOD in the sample solution is 0.636 x 10.75 = 6.84 U/ml.

Therefore, the SOD activity in 1 ml of rat blood can be calculated by using the dilution rate during the extraction process.
(15 / 0.5) x (11.5 / 10) x 6.84 = 236 U/ml


Results and Discussion:

Method 1:
The SOD activities of rat erythrocytes determined by using the cytocrhome C assay and WST assay were calculated and plotted as shown in Fig. 1.

Fig.1: Relationship between SOD activities in rat erythrocytes obtained using SOD Assay Kit-WST and cytochrome C methods




The correlation coefficient between these two assays (r) was 0.992 (n=12), and a high linearity was observed. From the slopes, the WST assay was 78 times more sensitive than the cytochrome C assay. This result was in agreement with the result obtained using Method 2, as indicated below. These results show that SOD Assay Kit-WST is useful for the SOD activity determination in biological samples.


Comparison of SOD activities determined using cytochrome C and WST assays

Calculation method of the unit of standard SOD:

Since 1 U is defined as the amount of protein in 3 ml solution that establishes 50% inhibition, we calculated 1 U as the amount of protein in 1 well (240 ul) that establishes 50% inhibition.

Cytochrome C Method:
Relative activity = 1 mg protein / (IC50 x 3 (ml))

Since IC₅₀ was 0.173 ug/ml in the assay solution, the following equation was applied.
1 / (0.173 x 3 / 1000) = 1927 U/mg protein

WST Method:
Relative activity = 1 mg protein / (IC50 x 0.24 (ml))

Since IC₅₀ was 0.0275 ug/ml in the assay solution, the following equation was applied.
1 / (0.0275 x 0.24 / 1000) = 152000 U/mg protein



Method 2:
The SOD activities of rat erythrocytes were determined by using the cytochrome C assay and WST assay. The SOD activities were calculated and plotted, as shown in Fig. 2.

Fig.2: Relationship between the values of SOD activities in rat erythrocytes obtained using SOD Assay Kit-WST and cytochrome C methods




The correlation coefficient between these two assays (r) was 0.903 (n=12), and a high linearity was observed as shown in Fig. 1. The units of the sample solution using the two methods were in the range of 150 U and 300 U, and the sensitivities of cytochrome C assay and WST assay were almost the same. These results show that SOD Assay Kit-WST is useful for the SOD activity determination in biological samples.


Comparison of Calculation Methods:
The WST assay and cytochrome C assay were compared using the two above-mentioned calculation methods. When Method 1 was used, the value determined using the WST assay was about 78 times higher than that of the cytochrome C assay. When Method 2 was used, however, the values derived using the WST assay and cytochrome C assay were almost the same. Therefore, Method 2 was determined to be the appropriate method.




Author:
Hiroyuki UKEDA, Ph.D.
Department of Agriculture
Khochi University
200 Mononobe Otsu
Nangoku-city, Khochi 783-8502
Japan

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