Purification of peroxidase enzyme from different radishes for preparation of elisa kit

Hoda Sotoudeh,1,* hossein amini ,2 Fatemeh mirkhani ,3 mohammad arjmand ,4 Sedigheh sadeghi ,5 zahra zamani ,6

1. Pasteur Institute of Iran
2. Pasteur Institute of Iran
3. Pasteur Institute of Iran
4. Pasteur Institute of Iran
5. Pasteur Institute of Iran
6. Pasteur Institute of Iran



Peroxidases are used widely in medical diagnostic kits and research applications. plant peroxidases (ec are heme containing oxidoreductases enzymes that oxidize a variety of organic and inorganic compounds using hydrogen peroxide. this enzyme is one of the glycoprotein hemoprotein that the prostatic group contains protopurephyrin fe ix. in the structure of this enzyme there is a calcium atom that is essential for the third protein structure. this enzyme has many applications in the field of immunoassay labroatory testing in the eliza technique for measuring hormones including thyroxin, insulin, hcg, estrogen, progesterone and toxin bacteria in addition to detecting tissue antigens and activating macrophages against cancer tumors in types of pathology and hematology and immunology are used. horseradish root is commonly used as a source of peroxidase as it is a rich source of this enzyme. turnip roots and horseradish roots are part of the brassicaceae family are also a good source of this enzyme. a comparative study was carried out on peroxidase enzyme was purified from two different sources of horse radish and turnip roots.


Horseradish and turnip roots were obtained from the local market. 620g of each were thoroughly washed with distilled water and cut down into small pieces and homogenized in a blender with potassium phosphate buffer ph 7.5. three precipitation steps were carried out using ammonium sulfate followed by ion exchange chromatography using both deae- cellulose (fig 1) and cm- cellulose columns (fig 2). activity of enzyme was carried out after each step at 420 nm using pyrogallol oxidation test. protein concentration was measured using photometric method at 280 nm. both purity and molecular weight were estimated using sds-page and silver staining.


The molecular weight of the protein purified by the sds-page method was determined with a concentration of 10% gel by coloring the silver nitrate at about 44 kda (fig 3). the enzyme activity for horseradish root 22.01 u/ml, the amount of protein concentration 0.124 mg/ml, specific activity 177.5 u/mg and its yield 51.23% was obtained. the activity of turnip root peroxidase 20.17 u/ml, the amount of protein concentration 0.1023 mg/ml, specific activity 197.165 u/mg and its yield 54.62% was acquired (table 1).


Purification of peroxidase enzyme in horseradish and turnip has been reported from horseradish and turnip roots. the calculated results have shown that the fractions with the highest enzyme activity after dialysis was passed through cm-cellulose column for horseradish and turnip roots peroxidase. it seems that the specific activity of the turnip is more than horseradish roots.


Peroxidase enzyme- purification- horseradish- turnip