PEROXIDASE from Horseradish

PEO-701

Donor:hydrogen-peroxidase oxidoreductase (EC 1.11.1.7)

Donor + H₂O₂ ► Oxidized donor + 2H₂O

PREPARATION and SPECIFICATION
Appearance:	Reddish-brown amorphous powder, lyophilized
Activity:	Grade VII 60 Purpurogallin U/mg-solid or more (Rz ≥ 0.6)

PROPERTIES
Stability:	Stable at -20°C for at least One year
Molecular weight :	approx. 40,000 (by gel filtration)
Structure:	Glycoprotein with one mole of protohaemin IX
Inhibitors:	Cyanide, sulfide, fluoride, azide
Optimum pH :	8.0(Fig.2)
pH Stability :	5.0-10.0(Fig.3)
Thermal stability :	below 60°C(Fig.4)

APPLICATIONS

This enzyme is useful for enzymatic determination of H₂O₂ in clinical analysis.

ASSAY

Principle:

peroxidase

2Pyrogallol+3H₂O₂ ► Purpurogallin+5H₂O+CO₂

The appearance of Purpurogallin is measured at 420nm by spectrophotometry.

Unit definition:

One Purpurogallin unit causes the formation of one milligram of purpurogallin in 20 seconds under the
conditions described below.

Method:

Reagents
A. Pyrogallol solution:	5% (W/V) (Should be prepared fresh)
B. H₂O₂ solution:	0.147M[Dilute 1.67ml of 30% (W/V) H₂O₂ to 100ml with H₂O] (Should be prepared fresh)
C. Phosphate buffer, pH6.0:	0.1M
D. H₂SO₄ solution:	2.0N

Procedure

Concentration in assay mixture
Phosphate buffer	15 mM
Pyrogallol	40 mM
H₂O₂	7.4mM

1. Prepare the following reaction mixture in a test tube (32ϕ ×200mm) and equilibrate at 20°C for about 5 minutes.

14.0ml H₂O
2.0ml Pyrogallol solution (A)
1.0ml H₂O₂ solution (B)
2.0ml Phosphate buffer, pH6.0 (C)

2. Add 1.0ml of the enzyme solution* and mix.

3. After exactly 20 seconds at 20°C, add 1.0ml of 2.0 N H₂SO₄ solution (D) to stop the reaction.

4. Extract the produced purogallin from the above stopped reaction mixture in five times with 15ml
portions of ether and fill up the combined ether extracts to 100ml with fresh ether.

5. Measure the optical density at 420nm against water (OD test).

At the same time, prepare the blank by first mixing the reaction with 1.0ml of 2.0N H₂SO₄
solution (D) after 20 sec-incubation at 20°C, followed by the addition of the enzyme solution and
extracting with ether by the same procedure as the test (OD blank).

* Dissolve the enzyme preparation in ice-cold 0.1M phosphate buffer, pH6.0 (C), dilute to 3.0-6.0
purpurogallin U/ml with the same buffer and store on ice.

Calculation

Activity** can be calculated by using the following formula :

ΔOD (OD test-OD blank) × Vt × df

Volume activity (U/ml) = = ΔOD×8.547×df

0.117×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vs: : Sample volume (1.0ml)
0.117: : Optical density at 420 nm corresponding to 1mg% of Purpurogallin in ether.
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)
**One purpurogallin unit is equivalent to 13.5 international units determined with o-dianisidine at 25°C.

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