Superoxide Dismutase (SOD) can catalyze the disproportionation reaction of superoxide anions, generating hydrogen peroxide (H2O2) and oxygen (O2). It is an important antioxidant enzyme in biological systems. Currently, there are several methods for measuring SOD activity, among which the NBT (Nitroblue Tetrazolium) method is widely used due to its ease of use. However, the methafulvin dye produced by the NBT method has poor water solubility and is prone to interact with the reduced xanthine oxidase, resulting in an inhibition rate that does not reach 100%, etc., which affects the sensitivity and accuracy of the detection; the Cytochrome C method is also a commonly used method for detecting SOD activity, but Cytochrome C has high oxidation activity and is easily interfered by reducing agents in the sample. Additionally, this method requires continuous measurement of absorbance values, resulting in relatively low detection sensitivity for SOD and is not suitable for the detection of large sample volumes.

The currently more advanced methods for measuring SOD include WST-1 method and WST-8 method. Among them, the WST-8 method is more stable and has higher sensitivity than the WST-1 method.

WST-8 can react with the superoxide anion (O2.-) produced by xanthine oxidase (XO) to generate water-soluble formazan dye. Since SOD can catalyze the disproportionation of superoxide anion, this reaction step can be inhibited by SOD. Therefore, the activity of SOD is negatively correlated with the amount of formazan dye produced. Thus, the enzymatic activity of SOD can be calculated by performing a colorimetric analysis of the WST-8 product.

Fig. 1 Detection Principle of WST-8 Method

Material and regents:

SOD sample preparation solution, SOD detection buffer solution, WST-8, enzyme solution, reaction initiation solution, 96-well plate、Microplate reader

Procedures:

1. Preparation

a. Preparation of WST-8/enzyme working solution: Prepare an appropriate amount of WST-8/enzyme working solution according to a volume of 160 μl for each reaction. Mix 151 μl of SOD detection buffer, 8 μl of WST-8, and 1 μl of enzyme solution evenly, and you can prepare 160 μl of WST-8/enzyme working solution. Prepare an appropriate amount of WST-8/enzyme working solution based on the number of samples to be tested (including standards). The specific preparation method can be referred to the following table. The prepared WST-8/enzyme working solution should be stored at 4°C or on ice and can be used on the same day. However, it is recommended to prepare and use it immediately.

Calculate based on the detection of 10 reactions:

b. Preparation of the reaction initiation working solution: Dissolve the reaction initiation solution (40X) in the reagent kit and mix well. Dilute it according to the ratio of 39μl SOD detection buffer for every 1μl reaction initiation solution (40X) and mix well to obtain the reaction initiation working solution. Prepare an appropriate amount of the reaction initiation working solution based on the quantity of the samples to be tested (including standards). Store the prepared reaction initiation working solution at 4°C or on ice. It can be used on the same day, but it is recommended to prepare and use it immediately.

2. Sample determination:

a. Refer to the table below to set up the sample wells and various blank control wells using a 96-well plate. Add the test sample and other various solutions in sequence according to the table. After adding the reaction initiation working solution, mix well. Note: Once the reaction initiation working solution is added, the reaction will start immediately. It is possible to operate at low temperature or use an aspirator to reduce the error caused by the different timing of adding the reaction initiation working solution in each well.

b. Incubate at 37°C for 30 minutes. Note: There is no significant difference in SOD activity detected after incubation for 25 to 35 minutes. However, to ensure consistency of the test results, it is recommended to incubate for 30 minutes.

c. Measure the absorbance at 450nm. If there is no 450nm filter, a 420-480nm filter can be used. You can choose to set 600nm (or above 600nm, such as 650nm) as the reference wavelength (also known as the reference wavelength). The absorbance reading at 450nm can be subtracted from the absorbance reading at the reference wavelength to obtain the actual measurement reading.

Fig. 2 SOD enzyme activity assay

We conducted relevant tests on the purified SOD enzyme solution. According to the SOD enzyme activity analysis chart, the residual superoxide anion concentration in the blank control group 1 (Blank1) was the highest, indicating that free radicals were abundant in the absence of enzyme intervention. While blank control group 2 (Blank 2) had the lowest superoxide anion concentration because it did not contain the reaction initiation solution, which confirmed the correctness of our detection system. At the same time, the residual free radical levels in the two experimental groups (SOD1-500mM, SOD2-500mM) were significantly reduced, suggesting that the purified SOD enzymes obtained have efficient scavenging activity.

Catalase is widely distributed. In the liver, kidneys and red blood cells, the level of catalase is extremely high, and it is the main site for removing hydrogen peroxide, which can cause oxidative damage.

The detection methods for hydrogen peroxide (H₂O₂) mainly include chemical colorimetry, fluorescence method, electrochemical method, enzyme-linked method and chemiluminescence method, etc. Chemical colorimetry uses the reaction of H₂O₂ with a color-developing reagent to produce colored products, which is quantified using a spectrophotometer. It is simple to operate and has low cost; fluorescence method has high sensitivity and is suitable for dynamic imaging; electrochemical method has a fast response and is suitable for on-site detection; enzyme-linked method has strong selectivity but requires high stability of the enzyme; chemiluminescence method has extremely high sensitivity and is suitable for ultra-trace analysis. In summary, chemical colorimetry still has significant advantages in laboratory and industrial site detection due to its simplicity, low cost and low equipment requirements.

The activity of catalase can also be determined by measuring A240 using a UV spectrophotometer. However, proteins or other components have relatively strong absorption around A240, which can cause serious interference to the measurement. Therefore, using the UV method to determine the activity of catalase is more suitable for purified catalase. This testing method detects the red product produced by the oxidation of the chromogenic substrate by hydrogen peroxide under the catalysis of peroxidase by measuring A520. It is less affected by interfering factors and has high detection sensitivity. It can detect as low as 1 U/ml of catalase.

When hydrogen peroxide is relatively abundant, catalase can catalyze the production of water and oxygen from hydrogen peroxide. The remaining hydrogen peroxide can be oxidized by peroxidase (Peroxidase) to produce a red product, N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine, with a maximum absorption wavelength of 520nm. Using hydrogen peroxide standards, a standard curve can be made. This allows the calculation of how much hydrogen peroxide has been catalyzed by the hydrogen peroxide enzyme in the sample to convert into water and oxygen within a unit time and a unit volume, thereby enabling the calculation of the enzyme activity of the hydrogen peroxide enzyme in the sample.

Material and regents:

Hydrogen peroxide enzyme detection buffer

Hydrogen peroxide (1M)

Hydrogen peroxide enzyme reaction termination solution

Color development substrate

Hydroperoxidase

Spectrophotometer

Microplate

Procedures:

1. Preparations:

a. Prepare a 5 mM hydrogen peroxide solution. Prepare a 5 mM hydrogen peroxide solution based on the actual hydrogen peroxide concentration obtained during the measurement.

b. Prepare the color development working solution. Dissolve the color development substrate on an ice bath, aliquot it appropriately for use, and avoid repeated freezing and thawing as much as possible. Other reagents should be placed on the ice bath for standby. Take an appropriate amount of peroxidase, dilute it with the color development substrate at a ratio of 1:1000, and prepare the color development working solution. For example, take 5 μl of peroxidase and add 5 ml of the color development substrate, mix well to obtain 5 ml of the color development working solution.

2. Standard curve determination

a. Take 0, 12.5, 25, 50 or 75 microliters of the prepared 5 mM hydrogen peroxide solution and add them to 1.5 ml or 0.5 ml plastic centrifuge tubes respectively. Add hydrogen peroxidase detection buffer to reach a final volume of 100 microliters. Mix well. At this time, the hydrogen peroxide solution concentrations are 0, 0.625, 1.25, 2.5, and 3.75 mM respectively. If necessary, higher concentration hydrogen peroxide standard solutions can be set.

b. Take 4 microliters each and add them to one well of the 96-well plate. Add 200 μl of the color development working solution. Incubate at 25°C for at least 15 minutes. However, the incubation time should not exceed 45 minutes. (Note: This step can be carried out simultaneously with the last step of the sample determination procedure.)

3. Sample analysis

a. Refer to the above table. Take x microliters (0 - 40 microliters) of the sample and transfer it to a 1.5 ml plastic centrifuge tube. Add hydrogen peroxidase detection buffer to a volume of 40 microliters (i.e., add 40 - x microliters of hydrogen peroxidase detection buffer), and mix well. Then add 10 microliters of 250 mM hydrogen peroxide solution and mix rapidly with a pipette. Refer to Table 1. The reaction should be carried out at 25°C for 1 - 5 minutes.

b. Add 450 microliters of hydrogen peroxidase reaction termination solution, invert and mix or vortex to terminate the reaction. These steps should be completed within 15 minutes after terminating the reaction.

c. In a clean plastic centrifuge tube, add 40 microliters of hydrogen peroxidase detection buffer, then add 10 microliters of the above-mentioned terminated and mixed reaction system, and mix well.

d. Take 10 microliters from the 50-microliter system and add it to one well of a 96-well plate. Add 200 microliters of the color development working solution.

e. Incubate at 25°C for at least 15 minutes, but the incubation time should not exceed 45 minutes.

Fig. 3 CAT enzyme activity assay

Superoxide anion free radicals, as a type of free radical produced during the metabolic process of organisms, can attack biological macromolecules such as lipids, proteins, nucleic acids, and polyunsaturated fatty acids, causing them to link or break, thereby damaging the structure and function of cells. They are closely related to the aging and diseases of the organism. Research on eliminating superoxide anion free radicals has received widespread attention.

Ion reactions produce measurable signals, which are easy to operate and highly sensitive; chemiluminescence method has high sensitivity and is suitable for trace detection; electrochemical methods can achieve real-time dynamic monitoring, but they require higher electrode materials. Different methods need to be balanced in terms of sensitivity, selectivity and ease of operation. The choice should be determined according to specific experimental requirements.

The AP-TEMED system generates superoxide anions, which react with hydroxylamine hydrochloride to form NO2-. NO2- reacts with p-amino benzenesulfonic acid and α-naphthylamine to form a red azo compound, which has a characteristic absorption peak at 530nm. The scavenging ability of the sample for superoxide anions is negatively correlated with the absorbance value at 530nm.

Material and regents:

Distilled water, micro plate analyzer, low-temperature centrifuge, constant temperature water bath, pipette, 96-well plate, cell sonicator

Procedures：

Sample preparation:

Tissue and cell samples should be pre-treated for disruption and centrifugation, and the supernatant obtained should be used for testing. Other liquid samples can be directly tested.

Procedure：

Allow the microplate reader to preheat for more than 30 minutes, and adjust the wavelength to 530nm.

Configure and conduct the experiment according to the table below:

Mix thoroughly, allow to color at 37 degrees Celsius for 20 minutes, and then measure the absorbance values of the blank tube and the test tube at 530nm.

Calculation of the scavenging ability of superoxide anions

Scavenging rate of superoxide anions (%): (A blank - A measurement) ÷ A blank * 100%

Fig. 4 Superoxide anion scavenging assay

A three-factor, three-level orthogonal experiment was conducted to systematically investigate the effects of SOD/CAT ratio, temperature, and pH on free radical scavenging efficiency. The results demonstrated that ​temperature was the most influential factor​ (range R = 74.38), with significantly higher scavenging efficiency observed at 25°C compared to 37°C and 45°C. Under high temperature (45°C), a substantial decrease in efficiency occurred, occasionally yielding negative values, indicating pronounced thermal inhibition of antioxidant enzyme activity. ​pH was identified as the second most critical factor​ (R = 13.56), where alkaline conditions (pH 8.0) favored enhanced scavenging performance, while neutral pH (7.0) resulted in the lowest efficiency. ​The SOD/CAT ratio had the least impact​ (R = 11.03). A ratio of 1:1 not only achieved the highest scavenging efficiency but also exhibited the best experimental reproducibility. In contrast, a ratio of 0.5:1 led to considerable variability and frequent negative values, indicating poor stability, whereas a ratio of 2:1 showed moderate performance. The ​optimal parameters were determined as follows: SOD/CAT ratio of 1:1, temperature of 25°C, and pH of 8.0. Under these conditions, the system achieved maximal radical scavenging efficacy. These findings provide a robust experimental basis and theoretical support for parameter optimization and the design of highly efficient antioxidant enzyme systems.

Reactive oxygen species (ROS) mainly include hydroxyl radicals, superoxide radicals and hydrogen peroxide. During the normal physiological metabolism of cells or tissues, reactive oxygen is produced. At the same time, some environmental factors such as ultraviolet radiation, gamma ray irradiation, smoking, and environmental pollution can also induce the production of reactive oxygen. After the generation of reactive oxygen, it can cause oxidative damage to lipids, proteins and DNA within cells, trigger oxidative stress (Oxidative stress), and subsequently lead to various tumors, atherosclerosis, rheumatoid arthritis, diabetes, liver damage, and central nervous system diseases. There are various antioxidants in the body, including antioxidant macromolecules, small molecules and enzymes, which can remove various reactive oxygen produced in the body to prevent the generation of oxidative stress (oxidative stress) induced by reactive oxygen. The total level of various antioxidant macromolecules, small molecules and enzymes in a system reflects the total antioxidant capacity of that system. Therefore, the determination of the total antioxidant capacity in various body fluids such as plasma, serum, urine, saliva, etc., and in cell or tissue lysates has very important biological significance.

The detection of the total antioxidant capacity of plant or herbal extract solutions, or various antioxidant solution solutions can be used to detect the strength of the antioxidant capacity of various solutions and can be used to screen drugs with strong antioxidant capacity.

ABTS⁺ radical (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) cationic radical) is a commonly used artificial radical and is widely employed in the determination of antioxidant activity. The ABTS⁺ radical has the characteristics of good stability, distinct solution color (with a blue-green absorption peak at approximately 734 nm) and ease of generation, making it an ideal model for evaluating the scavenging ability of antioxidants. During the detection process, the antioxidant reduces the ABTS⁺ radical, causing the color to gradually fade. The degree of absorbance reduction can be measured using a spectrophotometer, thereby calculating the antioxidant capacity.

Total Antioxidant Capacity Detection (ABTS Rapid Method), namely Total Antioxidant Capacity Assay Kit with a Rapid ABTS method, abbreviated as T-AOC Assay Kit, is a kit that uses 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS)) as the chromogenic agent. It can rapidly detect the total antioxidant capacity of various body fluids such as plasma, serum, saliva, urine, as well as lysates of cells or tissues, plant or herbal extract solutions, or various antioxidant solutions. The operation is simple and fast and is widely used in the evaluation of antioxidant activity of food, medicine and biological samples.

The principle of determining total antioxidant capacity by the ABTS method is shown in Figure 1. ABTS is oxidized into the green ABTS˙+ under the action of an appropriate oxidant. When an antioxidant is present, the production of ABTS˙+ is inhibited. The absorbance of ABTS˙+ at 414nm or 734nm can be measured to determine and calculate the total antioxidant capacity of the sample. Trolox is a similar substance to vitamin E and has a similar antioxidant capacity to vitamin E. It is used as a reference for the total antioxidant capacity of other antioxidants. For example, the total antioxidant capacity of Trolox is 1. At the same concentration, the antioxidant capacity of other substances is expressed as the multiple of its antioxidant capacity compared to Trolox.

Fig. 5 Schematic diagram of the principle of total antioxidant capacity detection using the ABTS method.

Material and regents:

Detection buffer, ABTS solution, hydrogen peroxide solution, peroxidase, Trolox solution (10 mM), microplate reader, 96-well plate, hydrogen peroxide

Procedures：

1. Dilution of hydrogen peroxide solution and peroxidase, as well as preparation of the ABTS working solution:

a. Dilute the hydrogen peroxide solution 1000 times with double-distilled water. For example, dissolve 10 microliters of hydrogen peroxide and add it to 9.99 ml of double-distilled water, then mix well to obtain a 1/1000 hydrogen peroxide solution.

b. According to the quantity of the sample to be tested, dilute an appropriate amount of peroxidase with the detection buffer solution 10 times. For example, take 20 microliters of peroxidase and add it to 180 microliters of the detection buffer solution, then mix well to prepare a 200-microliter peroxidase working solution. The peroxidase working solution should be freshly prepared and used.

c. Refer to the table below to prepare an appropriate amount of ABTS working solution based on the quantity of the sample to be determined (including the standard curve): For 10 reactions as an example

2. Preparation of the test samples:

a. Preparation of serum, plasma, saliva or urine samples:

Each sample of serum, plasma, saliva or urine requires 10 microliters and can be directly used for the test.

b. The supernatant obtained after extraction and lysis of cell or tissue samples can be used for the test.

c. Preparation of other samples:

Extracted solutions from plants or Chinese herbal medicines can be directly used for the test. It should be noted that the color of the sample itself will not interfere with the test. Other types of liquid samples can also be used for the test.

3. Preparation for standard curve determination:

Prepare the dilution solutions of the standards using distilled water or the sample. For serum, plasma, saliva or urine samples, directly dilute the standards with distilled water or PBS. For cell or tissue samples, dilute the standards using the solution for cell or tissue homogenate. For other samples, dilute the standards with the sample preparation solution. Dilute the 10 mM Trolox standard solution to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM.

4. Determination of total antioxidant capacity:

a. Add 20 microliters of the peroxidase working solution to each detection well of the 96-well plate.

b. Add 10 microliters of distilled water or an appropriate solution such as PBS to the blank control well; add 10 microliters of various concentrations of Trolox standard solution to the standard curve detection well; add 10 microliters of various samples to the sample detection well. Mix gently.

c. Add 170 microliters of ABTS working solution to each well and mix gently.

d. Incubate at room temperature for 6 minutes and measure A414. If it is difficult to measure A414, the measurement can be conducted within the range of 405-425 nm.

Fig. 6 ABTS⁺ free radical scavenging assay

Based on a three-factor, three-level orthogonal experimental design, this study systematically evaluated how the SOD/CAT ratio, temperature, and pH affect antioxidant capacity, measured by the Trolox equivalent antioxidant capacity (TEAC) assay. By analyzing the mean TEAC values and comparing their ranges for each factor, we determined that temperature has the greatest impact on antioxidant activity, followed by pH, while the SOD/CAT ratio plays a smaller but still meaningful role.

Notably, antioxidant capacity at 25°C was significantly higher than at 37°C and 45°C, and pH levels of 7.0 and 8.0 outperformed pH 6.0, demonstrating that lower temperatures and neutral-to-alkaline conditions markedly enhance antioxidant efficacy. Regarding the SOD/CAT ratio, although the highest TEAC value occurred at a 2:1 ratio, the increase over the 1:1 ratio was modest (range ≈ 0.1), indicating both ratios effectively contribute to antioxidant activity with limited variation.

Importantly, the optimal conditions were identified as an SOD/CAT ratio of 2:1, temperature of 25°C, and pH 7.0, under which the TEAC value peaked at 0.6519. This provides a clear, evidence-based guideline for designing experiments and optimizing processes to maximize antioxidant performance.

Overall, this work highlights the critical need for precise temperature and pH control to achieve superior antioxidant capacity, while offering flexibility in selecting the SOD/CAT ratio according to practical needs. These insights represent significant progress in understanding and harnessing the factors that govern antioxidant activity, paving the way for more effective applications.

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