Peroxidase(Horseradish)HRP

Introduction

Horseradish peroxidase (HRP, EC 1.11.1.7) is a glycoprotein composed of an enzyme protein and iron porphyrin. This enzyme consists of multiple isoenzymes, with a molecular weight of 40 kDa and an isoelectric point ranging from pH 3.0 to 9.0. The cofactor and enzyme protein exhibit maximum absorption spectra at 403 nm and 275 nm, respectively. Therefore, the RZ value (OD403nm/OD275nm ratio) is commonly used to indicate enzyme purity, with higher RZ values indicating greater purity.

Function

Lactoperoxidase is present in the mammary glands, salivary glands, and lacrimal glands of mammals, as well as in their respective secretions: milk, saliva, and tears. It is a common component of cow’s milk and human milk, and has been detected in the milk of all mammals studied to date. Lactoperoxidase was the first secreted peroxidase discovered and plays a crucial role in protecting the mammary glands of lactating females and the neonatal gut from pathogenic microorganisms, serving as part of the body’s defense system. Peroxidases in mammary, salivary, and lacrimal glands share similar chemical and immunological properties.

Applications

1. Horseradish peroxidase (HRP) is currently the most widely used labeling enzyme in ELISA tests, primarily due to its ease of preparation, relatively low cost, and stable properties. It exhibits heat and organic solvent resistance, with minimal loss of activity after conjugation with antigens or antibodies.
2. It is also commonly used in wastewater treatment. Industrial effluents often contain significant amounts of phenol, bisphenol A, and other aromatic amine compounds that cause substantial environmental pollution. Peroxidase can oxidize these pollutants as substrates into free radicals, which then polymerize into precipitate polymers, significantly reducing environmental contamination.
3. Recent research explores its use as a food additive. Being non-toxic and harmless, with easily controllable reaction temperatures, it has been studied for food preservation, detoxification, and detection applications.
4. Detection Applications: Can be combined with glucose oxidase for glucose content detection.

Synthesis Method
(1) Crush horseradish, extract, and centrifuge to obtain crude horseradish peroxidase extract;
(2) Ultrafiltrate the crude extract using a 70–100 kDa molecular weight cutoff ultrafiltration membrane to obtain ultrafiltrate;
(3) Concentrate the ultrafiltrate using ultrafiltration membranes with a molecular weight cutoff of 1–30 kDa to obtain horseradish peroxidase concentrate;
(4) Dehydrate the concentrate to obtain pure horseradish peroxidase.