Determination of CuZn-Superoxide Dismutase (SOD-1) Activity in Human Erythrocytes

Protocol for Determination of CuZn-Superoxide Dismutase (SOD-1) Activity in Human Erythrocytes

Principle of the assay:

CuZn-superoxide dismutase (SOD-1) activity was determined according to the method of Misra and Fridovich, based on the inhibition of adrenaline auto-oxidation under alkaline conditions. The rate of adrenochrome formation was monitored spectrophotometrically at 320 nm. One unit of SOD-1 activity was defined as the amount of enzyme causing 50% inhibition of adrenaline auto-oxidation and was expressed as U/g hemoglobin.

Preparation and storage of erythrocytes:

Whole blood was collected into K₂EDTA tubes and centrifuged at 1,500–2,000 × g for 10 min at room temperature to separate plasma, buffy coat, and erythrocytes. Plasma was carefully removed, and the buffy coat was discarded to minimize leukocyte and platelet contamination. The erythrocyte pellet was washed three times with isotonic saline (0.9% NaCl) using the same centrifugation conditions. After the final wash, the supernatant was carefully removed, and the washed erythrocytes were aliquoted and stored at −80 °C until analysis.

On the day of analysis, frozen erythrocytes were thawed at room temperature. Hemolysates were prepared immediately after thawing and used for hemoglobin determination, SOD-1 extraction, and enzymatic activity measurements without further storage.

Notes

Unless otherwise stated, all reagents were freshly prepared on the day of analysis. The adrenaline solution was prepared immediately before use because of its susceptibility to auto-oxidation. All measurements were performed in duplicate using a standard UV-Vis spectrophotometer equipped with 1-cm optical path-length cuvettes.

I. Reagents 

1. Adrenaline solution:

• Prepare 0.1 M hydrochloric acid by adding 78 μL of 39% HCl to deionized water and adjusting the final volume to 10 mL.

• Dissolve 16.488 mg of adrenaline in 10 mL of freshly prepared 0.1 M HCl.

2. Carbonate buffer (0.05 M, pH 10.2)

Prepare the buffer using:

• Na₂CO3 – 3.498 g

• NaHCO₃ – 1.428 g

• EDTA – 0.0372 g

        Dissolve all components in deionized water and adjust the final volume to 1 L. Adjust the pH to 10.2.

3. Chloroform–ethanol mixture (3:5, v/v) 

Prepare a chloroform–ethanol mixture by mixing:

• 3 mL chloroform

• 5 mL ethanol

II. Hemoglobin determination (Drabkin method)

• Preparation of Hemolysate I

Add 50 μL of packed washed erythrocytes to 150 μL of deionized water and mix thoroughly.

• Hemoglobin assay

Prepare the following reaction mixtures:

Test sample: 1.0 mL Drabkin reagent + 20 μL Hemolysate I

Standard: 1.0 mL cyanmethemoglobin standard solution

Blank: 1.0 mL Drabkin reagent

Mix all tubes thoroughly using a vortex mixer and incubate for 30 min at room temperature.

Measure absorbance of the test sample and the cyanmethemoglobin standard against the reagent blank at 540 nm.

• Hemoglobin calculation

1)   Hb [g/dL] = (A_B/A_S) × Cs × 0.0502

A_B - absorbance of the test sample

A_S - absorbance of the standard

C_s - concentration of the cyanmethemoglobin standard supplied with the assay

0,0502 - dilution factor

•    Preparation of Hemolysate II

Adjust the hemoglobin concentration of Hemolysate I to 5 g/dL.

• Calculate the required volume of deionized water using:

VH₂0 = (0.02 × Hb – 0,1) [mL]

where Hb is the hemoglobin concentration determined in Hemolysate I.

• Add 100 μL of Hemolysate I to the calculated volume of deionized water and mix thoroughly.

   

III. SOD-1 extraction

Pipette into a tube:

• 250 μL Hemolysate II 
• 200 μL chloroform–ethanol mixture (3:5) 
• 300 μL deionized water 

Mix vigorously using a vortex mixer for 1 min.

Centrifuge for 5 min at 3824 × g (6000 rpm) at 4 °C.

Following centrifugation, collect the upper aqueous phase, which contains SOD-1 activity.

The final volume of the aqueous extract is approximately 675 μL.

IV. Measurement of SOD-1 activity

All samples were incubated for 3 min at 30 °C before measurement.

Control

Control reaction:

• 1.45 mL carbonate buffer 
• 0.05 mL adrenaline solution  

Control blank:

• 1.50 mL carbonate buffer 

The increase in absorbance at 320 nm was monitored for 5 min against the corresponding blank.

Sample

Test reaction:

• 1.425 mL carbonate buffer 
• 25 μL SOD-1 extract 
• 50 μL adrenaline solution 

Sample blank:

• 1.475 mL carbonate buffer 
• 25 μL SOD-1 extract 

The increase in absorbance at 320 nm was monitored for 5 min against the corresponding blank.

V. Calculations

The percentage inhibition of adrenaline auto-oxidation by SOD-1 was calculated as:

                  % Inhibition = ((DE_K /min - DE_B /min)/ DE_K /min) × 100%

DE_B /min – rate of adrenaline oxidation in the presence of SOD-1

DE_K /min – rate of spontaneous adrenaline auto-oxidation (control)

One unit of SOD-1 activity was defined as the amount of enzyme producing 50% inhibition of adrenaline auto-oxidation:

[U] = % Inhibition/ 50

SOD-1 activity normalized to hemoglobin concentration was calculated as:

SOD-1 activity (U/g Hb) = (270.27 × U) / (V × HbII)

V – volume of SOD-1 extract used for the assay (mL)

HbII – hemoglobin concentration in Hemolysate II (g/dL)