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Send EmailPeroxidase, Horseradish Peroxidase, HRP, 9003-99-0
Peroxidase (Horseradish Peroxidase, HRP) is an oxidoreductase enzyme that catalyzes oxidation reactions between hydrogen peroxide (H₂O₂) and various electron donors (phenols, aromatic compounds, ABTS, etc.). Derived from horseradish (Armoracia rusticana) roots, this enzyme is most widely used in analytical biochemistry, particularly for signal generation in ELISA tests. It is also used in the food industry for flavor development and color stabilization, in biosensors for H₂O₂ detection, and in environmental technologies for the oxidative removal of dyes and phenolic compounds.
| Parameter | Information |
|---|---|
| Product Name | Peroxidase (Horseradish Peroxidase, HRP) |
| Other Names | Horseradish Peroxidase, HRP, Donor:Hydrogen Peroxide Oxidoreductase |
| CAS Number | 9003-99-0 |
| EC Number | 1.11.1.7 |
| Enzyme Class | Oxidoreductase |
| Source | Horseradish (Armoracia rusticana) roots or recombinant production (E. coli, yeast, etc.) |
| Appearance | Brownish to greenish-brown powder or liquid enzyme preparation |
| Odor | Slight characteristic odor |
| Physical State (20°C) | Solid (powder) or liquid |
| Type | Name |
|---|---|
| Common Names | Peroxidase, Horseradish Peroxidase, HRP |
| Systematic Name | Donor:Hydrogen Peroxide Oxidoreductase |
| EC Number | 1.11.1.7 |
| CAS Number | 9003-99-0 |
| HS Code | 3507.90 |
Peroxidase (HRP) is a monomeric glycoprotein with a molecular weight of approximately 44 kDa. It contains a heme group (iron protoporphyrin IX) in its active site, which is critical for catalytic activity. HRP consists of 308 amino acid residues and is stabilized by two disulfide bonds. The glycosylation rate is approximately 18-22%.
Structural Features:
Peroxidase (HRP)
|
Glycoprotein (~44 kDa)
|
Heme Cofactor (Fe-protoporphyrin IX)
|
Active Site + Substrate Binding Domain
|
H₂O₂ + Electron Donors (ABTS, Phenols, etc.)
| Feature | Description |
|---|---|
| Enzyme Type | Oxidoreductase |
| Cofactor | Heme group (iron protoporphyrin IX, Fe³⁺/Fe²⁺) |
| Molecular Weight | ~44 kDa (monomeric protein) |
| Amino Acid Count | 308 residues |
| Glycosylation | ~18-22% (N-linked glycans) |
| Isoelectric Point (pI) | 7.2 – 8.9 (isoenzyme dependent) |
| Substrate | H₂O₂ + electron donors (ABTS, TMB, DAB, phenols, guaiacol, etc.) |
| Optimal pH | 5.0 – 7.0 |
| Optimal Temperature | 30 – 40°C |
Peroxidase catalyzes oxidation reactions between hydrogen peroxide (H₂O₂) and various electron donors. The reaction begins with the oxidation of the enzyme's heme iron and proceeds through a two-step mechanism.
General Reaction:
H₂O₂ + AH₂ → 2H₂O + A
(Substrates: ABTS, TMB, Guaiacol, Phenols, etc.)
Reaction Mechanism (Steps):
HRP(Fe³⁺) + H₂O₂ → CI (HRP-Fe⁴⁺=O ·⁺) + H₂O (Compound I formation)
CI (Compound I) + AH₂ → CII (Compound II) + AH· (1st electron transfer)
CII (Compound II) + AH₂ → HRP(Fe³⁺) + AH· + H₂O (2nd electron transfer, regeneration)
Note: AH₂ represents the reduced electron donor, and AH· represents the oxidized radical product. The radical products may undergo dimerization, polymerization, or further oxidation reactions depending on the substrate.
| Isoenzyme | pI | Catalytic Efficiency | Description |
|---|---|---|---|
| HRP C | ~7.2 | High | Most common and best characterized isoenzyme |
| HRP A | ~5.5 | Moderate | Acidic isoenzyme |
| HRP B | ~8.9 | Low | Basic isoenzyme |
| HRP D | ~6.5 | Moderate | Intermediate isoenzyme |
Important Note: HRP C is the most common isoenzyme found in commercial preparations and is preferred for diagnostic applications such as ELISA.
| Property | Value |
|---|---|
| Appearance | Brownish to greenish-brown powder or liquid |
| Odor | Slight characteristic odor |
| Solubility | Soluble in water and dilute salt solutions |
| Optimal pH Range | 5.0 – 7.0 |
| Effective pH Range | 4.0 – 9.0 |
| Optimal Temperature | 30 – 40°C |
| Effective Temperature Range | 15 – 50°C |
| Inactivation Temperature | >60°C |
| Molecular Weight | ~44 kDa |
| Isoelectric Point (pI) | 7.2 – 8.9 (isoenzyme dependent) |
| Stability | Stable for 12 months when stored at 4-8°C |
| Parameter | Value |
|---|---|
| Activity Definition | One unit (U) is defined as the amount of enzyme required to oxidize 1 µmol of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) per minute at pH 7.0 and 25°C (ε₄₂₀ = 36 mM⁻¹cm⁻¹). |
| Typical Activity | Varies by manufacturer (typically 50 – 1,000 U/mg protein) |
| Substrate | H₂O₂ + electron donors (ABTS, TMB, DAB, guaiacol, phenols, o-dianisidine, etc.) |
| Reaction Products | Oxidized substrate products (colored or fluorescent products) + H₂O |
| Activators | Stabilizers (BSA, sucrose, glycerol) |
| Inhibitors | Sodium azide (NaN₃), heavy metals (Cu²⁺, Hg²⁺, Pb²⁺), sulfur-containing compounds, EDTA (high concentration), detergents (SDS) |
| Substrate | Maximum Absorbance (λmax) | Color | Description |
|---|---|---|---|
| TMB (3,3',5,5'-Tetramethylbenzidine) | 450 nm (acid) / 650 nm | Blue → Yellow | Most commonly used substrate in ELISA |
| ABTS | 420 nm | Green | Suitable for kinetic measurements |
| OPD (o-Phenylenediamine) | 490 nm | Orange-Yellow | Less used due to carcinogenic risk |
| DAB (Diaminobenzidine) | 450 nm | Brown | Used in histochemistry |
| Guaiacol | 470 nm | Brown | Classical activity measurement |
| Step | Description |
|---|---|
| 1 | HRP is conjugated to an antibody immobilized on the ELISA plate. |
| 2 | Substrate solution (H₂O₂ + TMB/ABTS) is added. |
| 3 | HRP uses H₂O₂ to oxidize the substrate. |
| 4 | A colored product is formed as a result of the oxidation reaction. |
| 5 | Color development is directly proportional to the amount of enzyme (and thus the target analyte). |
| 6 | Absorbance measurement (spectrophotometer) determines color intensity. |
| 7 | Target analyte concentration is calculated using a standard curve. |
| Application | Function |
|---|---|
| ELISA (Enzyme-Linked Immunosorbent Assay) | Detection and quantification of target analytes; HRP used for signal generation |
| Western Blotting | Protein detection using chromogenic or chemiluminescent substrates |
| Immunohistochemistry (IHC) | Antigen detection in tissue sections |
| Biosensors | Amperometric biosensors for H₂O₂ detection |
| Clinical Chemistry | Blood glucose, cholesterol, uric acid, etc. determinations (enzymatic methods) |
| Application | Function |
|---|---|
| Flavor Development | Oxidation of phenolic compounds for characteristic taste and aroma formation |
| Color Stabilization | Control of unwanted oxidative color changes in foods |
| Oxidative Reaction Control | H₂O₂ removal and regulation of oxidative degradation |
| Potato and Vegetable Processing | Control of enzymatic browning |
| Application | Function |
|---|---|
| Dye Removal | Oxidative degradation of dye compounds in wastewater |
| Phenolic Compound Removal | Oxidative polymerization and precipitation of phenol and derivatives |
| Wastewater Treatment | Removal of toxic organic substances from industrial wastewater |
| Application | Function |
|---|---|
| Biosensor Development | High-sensitivity biosensors for H₂O₂ detection |
| Enzyme Conjugation | Conjugation with antibodies, streptavidin, etc. |
| Chemiluminescence | High-sensitivity detection with chemiluminescent substrates (luminol) |
| Organic Synthesis | Selective oxidation reactions |
| Application | Recommended Dosage | Conditions |
|---|---|---|
| ELISA | 1:1,000 – 1:10,000 (conjugate dilution) | pH 7.0-7.4, 25-37°C |
| Western Blot | 1:1,000 – 1:5,000 (conjugate dilution) | pH 7.0-7.4, 25°C |
| Immunohistochemistry | 1:100 – 1:500 (conjugate dilution) | pH 7.0-7.4, 25-37°C |
| Food Processing | 0.001 – 0.01% (product basis) | pH 5.0-7.0, 20-40°C |
| Biosensor | Varies by application | pH 7.0, 25°C |
Note: Dosage should be optimized for each specific application through trial runs. For ELISA and other diagnostic applications, optimal conjugate dilution should be determined by titration experiments.
| Parameter | Recommendation |
|---|---|
| Dilution Buffer | PBS (Phosphate Buffered Saline), pH 7.0-7.4 + BSA (0.1-1.0%) |
| Substrate Preparation | TMB or ABTS should be prepared according to manufacturer instructions |
| Reaction Time | 5-30 minutes (at room temperature) |
| Reaction Stop | 1-2 M H₂SO₄ (for TMB) or SDS solution |
| Temperature | Room temperature (20-25°C) or 37°C (incubator) |
| Inactivation | Inactivated above 60°C; acid or base inactivation also possible |
| Factor | Effect |
|---|---|
| Temperature | Optimal 30-40°C; activity decreases above 50°C; >60°C inactivation |
| pH | Optimal pH 5.0-7.0; significant activity at pH 4.0-9.0 |
| Substrate Concentration | High H₂O₂ concentration can cause enzyme inhibition |
| Inhibitors | Sodium azide (NaN₃), heavy metals, sulfur-containing compounds, detergents |
| Buffer Ions | Different buffer systems may affect activity |
| Ionic Strength | High ionic strength may reduce activity |
| Protein Stabilizers | BSA, glycerol, sucrose increase stability |
| Parameter | Value |
|---|---|
| Acute Oral Toxicity (LD50, Rat) | > 2,000 mg/kg (low toxicity) |
| Acute Dermal Toxicity | Low |
| Acute Inhalation Toxicity | Powder form may cause respiratory sensitization |
| Skin Irritation | Mild irritant |
| Eye Irritation | May cause mild irritation |
| Skin Sensitization | May cause sensitization in sensitive individuals (protein-based) |
| Mutagenicity | Negative (Ames test) |
| Carcinogenicity | Not classified as a carcinogen |
| Reproductive Toxicity | Not classified as a reproductive toxin |
| Allergenicity | May cause allergic reactions in sensitive individuals (protein-based) |
| Hazard Class | Category | H-Statement |
|---|---|---|
| Respiratory Sensitization | Category 1 | H334 |
| Skin Sensitization | Category 1 | H317 |
| Water Hazard (WGK Germany) | 3 (severe hazard to water) | - |
Signal Word: WARNING
Hazard Pictograms: GHS07 (Exclamation Mark), GHS08 (Health)
Hazard Statements (H-Codes):
| Code | Statement |
|---|---|
| H317 | May cause an allergic skin reaction. |
| H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled. |
| Code | Statement |
|---|---|
| P261 | Avoid breathing dust/fume/gas/mist/vapors/spray. |
| P264 | Wash hands thoroughly after handling. |
| P271 | Use only outdoors or in a well-ventilated area. |
| P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
| P302+P352 | IF ON SKIN: Wash with plenty of soap and water. |
| P304+P340 | IF INHALED: Remove person to fresh air and keep comfortable for breathing. |
| P305+P351+P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present. |
| P342+P311 | If experiencing respiratory symptoms: Call a POISON CENTER/doctor. |
| P333+P313 | If skin irritation or rash occurs: Get medical advice/attention. |
| P337+P313 | If eye irritation persists: Get medical advice/attention. |
| P362+P364 | Take off contaminated clothing and wash it before reuse. |
| P501 | Dispose of contents/container in accordance with local regulations. |
| Route of Exposure | Action to Be Taken |
|---|---|
| Inhalation | Move to fresh air. If respiratory irritation persists, seek medical attention. |
| Skin Contact | Wash with plenty of soap and water. Remove contaminated clothing. If irritation persists, consult a physician. |
| Eye Contact | Rinse thoroughly with plenty of water for at least 15 minutes. Remove contact lenses. If irritation persists, seek medical attention. |
| Ingestion | Rinse mouth with water. Drink plenty of water. Do not induce vomiting. If large amount is swallowed, seek medical attention. |
| Parameter | Information |
|---|---|
| Fire Hazard | Combustible organic powder; may burn at high temperatures. |
| Hazards During Fire | Thermal decomposition produces toxic fumes including carbon monoxide, carbon dioxide, and nitrogen oxides. |
| Suitable Extinguishing Media | Water spray, CO₂, dry chemical powder, alcohol-resistant foam. |
| Special Protective Equipment | Self-contained breathing apparatus (SCBA), full protective clothing. |
| Parameter | Information |
|---|---|
| Personal Protection | Dust mask, protective goggles, gloves, coverall. |
| Ventilation | Increase ventilation; use local exhaust in confined spaces. |
| Containment | Cover spilled area to prevent dust dispersion. |
| Cleaning Methods | Collect dry using vacuum or careful sweeping. Avoid washing with water (soluble). |
| Environmental Precautions | Prevent entry into drains, sewers, and water bodies. |
| Waste Disposal | Dispose of in accordance with local regulations. |
| Parameter | Information |
|---|---|
| Storage Conditions | Store at 2-8°C (long-term) or -20°C (very long-term) in a cool, dry environment. |
| Short-Term Storage | Several weeks at 2-8°C (in solution, stabilized with BSA) |
| Long-Term Storage | -20°C recommended (years of stability in powder form) |
| Container Requirements | Keep tightly closed in original packaging. Protect from moisture and direct sunlight. |
| Materials to Avoid | Strong oxidizers, high heat, moisture, sodium azide (inhibitor) |
| Shelf Life | 12 months (in unopened original packaging under proper storage) |
| Stability Note | Stable for 12 months when stored at 4-8°C; much longer stability at -20°C. Stability in solution can be enhanced with BSA and glycerol. |
| Packaging Options | Aluminum foil bags, plastic containers, vacuum packaging. |
| Packaging Type | Quantity | Material |
|---|---|---|
| Aluminum Foil Bag | 1 g, 5 g, 10 g | Laminated aluminum (nitrogen atmosphere) |
| Plastic Container | 1 g, 5 g, 10 g | HDPE / Food-grade plastic |
| Glass Vial | 1 g, 5 g, 10 g | Amber glass (light protection) |
| Parameter | Information |
|---|---|
| UN Number | Not applicable (not classified as dangerous goods) |
| Hazard Class | Not classified as hazardous for transport |
| Packing Group | Not applicable |
| ADR/RID | Not regulated |
| IMDG Code | Not regulated |
| IATA (Air) | Not regulated |
| Marine Pollutant | No |
| Transport Temperature | Cold chain (2-8°C) or ambient (depending on product form); protect from moisture. |
| Region / Authority | Status |
|---|---|
| European Union | Approved as a food enzyme (EU 1332/2008). |
| USA (FDA) | GRAS (Generally Recognized as Safe) for food applications. |
| Turkey | Approved as a food processing aid (Turkish Food Codex). |
| EFSA | Re-evaluated and considered safe at current usage levels. |
| Kosher/Halal | Can be certified upon request (depending on source). |
| WGK Germany | 3 (severe hazard to water) |
| Parameter | Information |
|---|---|
| Aquatic Toxicity | Low toxicity to aquatic organisms. |
| Biodegradability | Readily biodegradable (protein-based). |
| Bioaccumulation | Not expected. |
| Water Hazard Class (WGK Germany) | 3 (severe hazard to water). |
| Waste Disposal | Dispose of in accordance with local regulations. Incineration is recommended for contaminated materials. |
| CAS Number | Enzyme | Description |
|---|---|---|
| 9003-99-0 | Peroxidase (HRP) | This CAS number is used for Horseradish Peroxidase. |
| 9001-05-2 | Catalase | This is a different enzyme (EC 1.11.1.6); should not be confused with peroxidase. |
Important Note: Catalase (CAS 9001-05-2) and peroxidase (CAS 9003-99-0) are different enzymes.
Catalase (EC 1.11.1.6): Decomposes H₂O₂ to water and oxygen (2H₂O₂ → 2H₂O + O₂)
Peroxidase (EC 1.11.1.7): Uses H₂O₂ to oxidize organic substrates (H₂O₂ + AH₂ → 2H₂O + A)
Verify the correct CAS number and EC number on the supplier's SDS.
| Property | Peroxidase (HRP) | Catalase | Glucose Oxidase | Laccase |
|---|---|---|---|---|
| Enzyme Class | Oxidoreductase | Oxidoreductase | Oxidoreductase | Oxidoreductase |
| EC Number | 1.11.1.7 | 1.11.1.6 | 1.1.3.4 | 1.10.3.2 |
| Cofactor | Heme (Fe) | Heme (Fe) | FAD | Cu |
| Substrate | H₂O₂ + AH₂ | H₂O₂ | Glucose + O₂ | Phenols + O₂ |
| Reaction | H₂O₂ + AH₂ → 2H₂O + A | 2H₂O₂ → 2H₂O + O₂ | Glucose → Gluconic Acid | Phenols → Quinones |
| Optimal pH | 5.0-7.0 | 7.0-8.0 | 5.5-7.0 | 4.0-6.0 |
| Optimal Temperature | 30-40°C | 20-30°C | 30-40°C | 25-55°C |
| Primary Application | ELISA, biosensor | H₂O₂ removal | Biosensor | Pulp, paper |
Q1: What is peroxidase (HRP) and what is it used for?
A: Peroxidase (Horseradish Peroxidase) is an oxidoreductase enzyme that catalyzes oxidation reactions between H₂O₂ and various electron donors. Its most common use is signal generation in ELISA tests. It is also used in the food industry for flavor and color control, in biosensors for H₂O₂ detection, and in environmental technologies.
Q2: What is the difference between peroxidase and catalase?
A: Peroxidase (CAS 9003-99-0, EC 1.11.1.7) uses H₂O₂ to oxidize organic substrates (H₂O₂ + AH₂ → 2H₂O + A). Catalase (CAS 9001-05-2, EC 1.11.1.6) decomposes H₂O₂ to water and oxygen (2H₂O₂ → 2H₂O + O₂). They are different enzymes with different applications.
Q3: What is the optimal pH and temperature for peroxidase?
A: The optimal pH is 5.0-7.0 and the optimal temperature is 30-40°C. The enzyme is inactivated above 60°C. For diagnostic applications such as ELISA, it is typically used at pH 7.0-7.4 and room temperature or 37°C.
Q4: Is peroxidase safe?
A: Yes. Peroxidase is considered safe for food applications. However, the powder form may cause respiratory or skin sensitization in sensitive individuals. Avoid inhaling the powder; wash with water in case of eye or skin contact.
Q5: How should peroxidase be stored?
A: Store at 2-8°C (long-term) or -20°C (very long-term). Solutions can be stabilized with BSA and glycerol. Keep in a cool, dry, light-protected environment in tightly closed original packaging. Stable for 12 months when stored at 4-8°C.
Q6: How does HRP work in ELISA?
A: In ELISA, HRP is conjugated to an antibody or streptavidin. When substrate solution (H₂O₂ + TMB/ABTS) is added, HRP oxidizes the substrate to produce a colored product. The color intensity is directly proportional to the amount of target analyte. The color is measured with a spectrophotometer and concentration is calculated using a standard curve.
Q7: What is the CAS number for peroxidase?
A: The CAS number for Horseradish Peroxidase (HRP) is 9003-99-0. Catalase (CAS 9001-05-2) is a different enzyme and should not be confused with peroxidase. Verify the correct CAS number on the supplier's SDS.
Q8: What inhibits peroxidase activity?
A: Sodium azide (NaN₃), heavy metals (Cu²⁺, Hg²⁺, Pb²⁺), sulfur-containing compounds, high concentrations of EDTA, and detergents (SDS) inhibit peroxidase activity. High H₂O₂ concentration can also cause enzyme inhibition.
| Property | Value |
|---|---|
| CAS Number | 9003-99-0 |
| EC Number | 1.11.1.7 |
| Enzyme Class | Oxidoreductase |
| Cofactor | Heme group (Fe-protoporphyrin IX) |
| Molecular Weight | ~44 kDa |
| Appearance | Brownish to greenish-brown powder or liquid |
| Solubility | Soluble in water |
| Optimal pH | 5.0 – 7.0 |
| Optimal Temperature | 30 – 40°C |
| Inactivation Temperature | >60°C |
| Substrate | H₂O₂ + electron donors (ABTS, TMB, DAB, phenols, etc.) |
| Primary Uses | ELISA, Western blot, immunohistochemistry, biosensors, food processing, environmental technologies |
| Shelf Life | 12 months (at 2-8°C, under proper storage) |
| UN Number | Not applicable |
| GHS Signal Word | WARNING |
| WGK Germany | 3 (severe hazard to water) |
CAS Number Verification: The CAS number for peroxidase is 9003-99-0. Catalase (CAS 9001-05-2) is a different enzyme and should not be confused with peroxidase. Catalase decomposes H₂O₂ to water + oxygen, while peroxidase uses H₂O₂ to oxidize organic substrates. Verify the correct CAS number and EC number on the supplier's SDS.
Respiratory Sensitization: Peroxidase powder may cause respiratory sensitization (H334) in susceptible individuals. Use appropriate dust control measures and respiratory protection. Avoid inhaling the powder.
Skin Sensitization: May cause allergic skin reactions (H317) in sensitive individuals. Use protective gloves. Wash with water in case of eye or skin contact.
Temperature Sensitivity: Peroxidase is heat-sensitive. Inactivated above 60°C. Pay attention to processing and storage temperatures.
Sodium Azide (NaN₃) Inhibition: Sodium azide is a strong inhibitor of peroxidase. Do not use sodium azide in storage buffers or working solutions.
Light Sensitivity: Peroxidase solutions are light-sensitive. Protect from light.
Substrate Selection: Different substrates (TMB, ABTS, OPD, DAB) provide different sensitivity and measurement wavelengths. Choose the appropriate substrate for the application.
Dosage Optimization: For ELISA and other diagnostic applications, optimal conjugate dilution should be determined by titration experiments.
Storage: Store at 2-8°C (long-term) or -20°C (very long-term). Keep powder form dry. Store in tightly closed original packaging. Protect from moisture and direct sunlight. Stability in solution can be enhanced with BSA (0.1-1.0%) and glycerol (30-50%).
Handling: Use dust mask, protective gloves, and goggles. Avoid dust formation. Ensure adequate ventilation. Use sodium azide-free buffers.
ELISA Application: Dilute conjugate in PBS (pH 7.0-7.4) + BSA (0.1-1.0%). Use TMB or ABTS substrate. Reaction time: 5-30 minutes. Stop reaction with 1-2 M H₂SO₄ or SDS. Measure absorbance at appropriate wavelength.
Food Application: Use at pH 5.0-7.0, temperature 20-40°C. Ensure controlled oxidation.
Quality Control: Monitor enzyme activity regularly. RZ (Reinheitszahl) value (A₄₀₃/A₂₇₅) is an indicator of purity.
Waste Management: Dispose of waste in accordance with local regulations. Do not release into water bodies.
Environmental Protection: Due to WGK 3 classification, prevent entry into drains, sewers, and water bodies.
This Technical Data Sheet (TDS) is for informational purposes only and is prepared based on available technical data. The user is solely responsible for determining the suitability of the product for their specific application and for complying with all local, national, and international regulations. For complete safety, storage, handling, transport, waste, and regulatory compliance information, the official Safety Data Sheet (SDS/MSDS) provided by the manufacturer/supplier must be consulted. This document does not substitute professional advice. Dosage and application conditions should be optimized through product-specific trials. The CAS number for peroxidase is 9003-99-0. Catalase (CAS 9001-05-2) is a different enzyme; do not confuse with peroxidase. Verify the correct CAS number and EC number on the supplier's SDS.