Peroxidase, Horseradish (POD, HRP, HRPO)

Blue Ice

44

Supplied as a lyophilized powder. The choice of solvent will depend on the intended application. The powdered enzymes are soluble in H2O or 0.1M phosphate buffer, pH 6 (10mg/ml).

Horseradish peroxidase (HRP) is isolated from horseradish roots (Amoracia rusticana) and belongs to the ferroprotoporphyrin group of peroxidases. HRP is a single chain polypeptide containing four disulfide bridges. It is a glycoprotein containing 18% carbohydrate. The carbohydrate composition consistsof galactose, arabinose, xylose, fucose, mannose, mannosamine, and galactosamine, depending upon the specific isozyme.2 Its molecular weight (~44kD) includes the polypeptide chain (33, 890 Daltons), hemin plus Ca2+ (approx. 700 Daltons), and carbo-hydrate (9400 Daltons).3 At least seven isozymes of HRP exist.2 The isoelectric point for horseradish Peroxidase isozymes ranges from 3-9. HRP readily combines with hydrogen peroxide (H2O2) and the resultant [HRP-H2O2] complex can oxidize a wide variety of chromogenic hydrogen donors. It can also utilize chemiluminescent substrates such as luminol and isoluminol and fluorogenic substrates such as tyramine, homovanillic acid, 4-hydroxyphenyl acetic acid.

The following compounds are inhibitors of horseradish peroxidase: sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfide, vanadate, paminobenzoic acid, Cd+2, Co+2, Cu+2, Fe+3, Mn+2, Ni+2, Pb+2. 4 The pH optimum of HRP is in the range of 6.0 to 6.5; activity at 7.5 is 84% of the maximum. The enzyme is most stable in the pH range of 5.0 to 9.0.5 Horseradish peroxidase is widely used as a label for immunoglobulins in many different immunochemistry leads to lower non-specific binding.6 Protocols describing the glutaraldehyde and periodate conjugation methodologies can be reviewed in Harlow, E. et al.

Source:
HRP, from horseradish roots (Amoracia rusticana)

RZ (Reinheitszahl):
Absorbance ratio of A403/A275. RZ=3.0.

Purpurogallin Activity (units per mg solid):
~250-330

Purpurogallin Unit Definition:
One unit will form 1.0mg purpurogallin from pyrogallol in 20 seconds at pH 6.0 at 20 °C. This unit is equivalent to approx. 18uM units/minute at 25 °C.

ABTS Unit Definition:
One unit will oxidize 1umole of 2, 2’-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)/minute at 25°C, pH 5.0.

ABTS Activity (units per mg solid):
~1000

Storage and Stability:
Lyophilized powder may be stored at -20°C. Stable for 12 months after receipt at -20°C. Reconstitute with sterile buffer or ddH2O. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Reconstituted product is stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.