Catalase (CAT) Activity Assay Kit (UV Colorimetric Method)

Cat. No.: H1508203

GRADE & PURITY BioReagent

Descripción general

Catalase (CAT), also known as hematase, is a conjugated enzyme that uses iron porphyrin as its prosthetic group. It is a tetrameric enzyme composed of four identical subunits, containing four molecules of heme as cofactors, with a molecular weight of approximately 24 kDa. CAT rapidly eliminates hydrogen peroxide, a toxic substance produced by cell metabolism, and works together with GSH‑Px to protect sulfhydryl enzymes, membrane proteins, and to dissociate hydrogen peroxide. The detection of this enzyme holds certain value for studying plant metabolic intensity as well as drought and disease resistance.

Detection Principle

Hydrogen peroxide (H₂O₂) in samples such as serum or plasma has strong absorption at 240 nm. Catalase decomposes H₂O₂, causing the absorbance of the test solution to decrease over time. The activity of catalase can be determined by measuring the rate of change in absorbance at 240 nm using a UV spectrophotometer.

Applicable Samples: Animal and plant tissues; Serum; Plasma; Cells; Bacteria

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 240 nm)

• Quartz cuvette

• Mortar or homogenizer, Centrifuge tubes, Low‑temperature centrifuge, Water bath or incubator

• Distilled water, Physiological saline

Procedure

1. Preparation of CAT Assay Buffer Working Solution

Dilute CAT Assay Buffer (2.5×) with distilled water at a ratio of 1:1.5 to obtain the CAT Assay Buffer Working Solution. Pre‑cool at 4°C for later use.

2. Preparation of 100 mM H₂O₂ Stock Solution

The H₂O₂ stock solution provided in this kit has an approximate H₂O₂ concentration of 1 M. Since hydrogen peroxide is not very stable, its actual concentration must be determined before use. Dilute the approximately 1 M H₂O₂ stock solution 100‑fold with CAT Assay Buffer Working Solution to obtain an approximately 10 mM H₂O₂ solution. Measure its absorbance at 240 nm (A₂₄₀) using a spectrophotometer (typically, a freshly prepared 10 mM H₂O₂ solution has an A₂₄₀ around 0.45, which may drop to around 0.42 after 3 months). Calculate the H₂O₂ concentration (mM) using the formula: H₂O₂ concentration (mM) = 22.94 × A₂₄₀. This gives the actual H₂O₂ concentration in the stock solution provided. Based on this actual concentration, prepare a 100 mM H₂O₂ stock solution accordingly.

3. Sample Preparation

3.1 Plant and Animal Samples

Take fresh plant or animal tissue (normal or under stress), wash clean, dry, cut into small pieces, and quickly weigh. Homogenize on ice using a tissue mass (g) to CAT Assay Buffer Working Solution volume (mL) ratio of 1:5‑10 (recommended: weigh about 0.1 g tissue and add 1 mL CAT Assay Buffer Working Solution). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for testing.

3.2 Plasma, Serum, and Urine Samples

Prepare plasma and serum using standard methods. Dilute 10‑fold with physiological saline, then they can be directly used for assay with this kit. Urine can usually be tested directly. Keep samples on ice.

3.3 Bacterial or Cultured Cell Samples

Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Based on a ratio of bacterial/cell count (10⁴) to CAT Assay Buffer Working Solution volume (mL) of 500‑1000:1 (recommended: add 1 mL CAT Assay Buffer Working Solution per 5 million bacteria or cells), disrupt the bacteria or cells by sonication (power 200 W, pulse 3 seconds, interval 10 seconds, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for testing.

4. CAT Sample Loading

Set up blank and test tubes according to the table below. Add solutions in the specified order, avoiding bubble formation. If the enzyme activity in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is best to set up replicate tubes for each sample.

Additive (mL)	Blank Tube	Test Tube Ⅰ	Test Tube Ⅱ
CAT Assay Buffer Working Solution	1.5	1.5	1.5
Test Sample (or extract)	0.2	0.2	0.2
Distilled Water	1.0	1.0	1.0

Boil the blank tube for 1 minute, then cool to 25°C. Preheat the test tubes to 25°C.

5. CAT Assay

Add 0.3 mL of 100 mM H₂O₂ Stock Solution. Start timing immediately after adding to each tube, and quickly pour the mixture into a quartz cuvette. Zero the instrument with distilled water. Measure the absorbance at 240 nm for each tube using the spectrophotometer, taking readings every 1 minute for a total of 4 readings. After all measurements are completed, calculate the enzyme activity.

6. Calculation of Results

6.1 Calculation of CAT Activity in Plant/Animal Tissues

(1) Based on sample mass

Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per gram of tissue at 25°C.

CAT [U/(g·min)] = (ΔA₂₄₀ × V_T × N) / (0.1 × V_S × t × W)

(2) Based on protein concentration

Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per milligram of tissue protein at 25°C.

CAT [U/(mg·min)] = (ΔA₂₄₀ × V_T × N) / (0.1 × V_S × t × Cpr)

6.2 Calculation of CAT Activity in Serum, Plasma, and Urine

Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per milliliter of liquid sample at 25°C.

CAT [U/(mL·min)] = (ΔA₂₄₀ × N) / (0.1 × V_S × t)

6.3 Calculation of CAT Activity in Bacteria/Cells

Unit definition: One unit of CAT activity is defined as the amount of enzyme that causes a decrease in absorbance of 0.1 per minute at 240 nm per 10⁴ bacterial or cell sample at 25°C.

CAT [U/(10⁴ cell·min)] = (ΔA₂₄₀ × V_T × N) / (0.1 × V_S × t × n)

Parameter Description:

ΔA₂₄₀ = A_Blank – (A_{Test I} + A_{Test II}) / 2

A_Blank: Absorbance of the blank

A_Test: The final, relatively stable absorbance of the test sample

V_T: Total volume of enzyme extraction solution (mL)

N: Dilution factor of the test sample before measurement

V_S: Volume of sample used in the assay (mL)

t: Reaction time from addition of H₂O₂ stock solution to the last reading (min)

W: Fresh weight of the sample (g)

n: Number of cells or bacteria, in units of 10⁴ (500~1000)

0.1: One enzyme activity unit corresponds to a decrease in A₂₄₀ of 0.1

Notes

1. Test samples should not contain CAT inhibitors, and repeated freeze‑thaw cycles should be avoided.

2. For absorbance measurements below 400 nm, quartz cuvettes should be used.

3. If precipitates or flocculants appear in the CAT Assay Buffer, warming in approximately 50°C water may help dissolve them. If flocculants persist, discard the buffer.

4. Intact red blood cells and undiluted hemolysate maintain stable catalase activity for up to 1 week at 4°C. CAT in diluted hemolysate is prone to inactivation.

5. Avoid hemolysis caused by freezing samples, as it can reduce catalase activity by 10‑15%.

6. Serum samples lose 64.7% activity at room temperature within 3 days, 10.5% at 4°C, but only 3.5% after 30 days at ‑20°C. Therefore, test samples should be stored at ‑20°C or ‑70°C.

7. If a large number of bubbles form in the reaction mixture, dilute the sample with distilled water before measurement.

8. For your safety and health, please wear a lab coat and disposable gloves during operation.

9. Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.

10. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

H1508203	Component	100T	Storage
H1508203A	H₂O₂ Stock Solution	5 mL	2-8℃. Store in the dark.
H1508203B	CAT Assay Buffer (2.5×)	250 mL×2	2-8℃