PEROXIDASE from Horseradish

PEO-131, 301, 302

Donor:hydrogen-peroxidase oxidoreductase (EC 1.11.1.7)

Donor + H₂O₂                      ► Oxidized donor + 2H₂O

PREPARATION and SPECIFICATION
PEO-301/302 are isolated from horseradish root in our own method. PEO-131 is chromatographically separated from the other isoenzymes using SP Sephadex C-50 by the modified method of Paul et al.1) The peroxidase fractions having equal RZ values (ca.3.3) are combined to generate the preparation. This preparation has an RZ value of ca.3.3 and is electrophoretically homogeneous. On the other hand, GradeIII is partially purified preparation.
Appearance:  Reddish-brown amorphous powder, lyophilized
Activity:  GradeⅠ 250 Purpurogallin U/mg-solid or more
(-131) (RZ ≥ 3.0, salt free)
GradeⅢ 110 Purpurogallin U/mg-solid or more
(-301) (RZ ≥ 2.0, containing approx. 30% of stabilizers)
GradeⅢ 180 Purpurogallin U/mg-solid or more
(-302) (RZ ≥ 2.0, salt free)
Contaminant:  Phosphatase ≤1.0×10⁻³% (GradeⅢ)
PROPERTIES
Stability:  Stable at -20°C for at least Two years (Fig.1,2,3)
Molecular weight :  approx. 40,000
Structure:  Glycoprotein with one mole of protohaemin IX ² ⁾
Inhibitors:  Cyanide, sulfide, fluoride, azide ³ ⁾
Optimum pH :  6.0-7.0(Fig.6)
Optimum temperature :  45°C(Fig.7)
pH Stability :  pH 5.0-10.0 (25°C, 20hr)(Fig.8)
Thermal stability :  below 50°C (pH 6.0, 10min)(Fig.9)
Effect of various chemicals :  (Table 1)

APPLICATIONS ⁴ ˜¹¹⁾

This enzyme is useful for enzymatic determination of H₂O₂ in clinical analysis. Especially, the highly purified preparation (Grade I) is useful as a protein tracer in histo-and cyto-chemistry and as a valuable experimental tool in hodological neurography. Also, the enzyme preparation has been used as an enzyme label in enzyme immunoassay. Grade III(-302) is suitable for dry chemistry. On the other hand, the enzymes contribute for the reduction of phehol in waste water.

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 purpurogallin 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 mixture with 1.0ml of 2.0 N H₂SO₄ solution (D) after 20 a 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, pH 6.0 (C), dilute to 3.0-6.0 pur purogallin 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)×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.

REFERENCES

  1. K.G.Paul and T.Stigbrand; Acta Chem.Scand., 24, 3607 (1970).
  2. L.M.Shannon et al.; J.Biol.Chem., 241, 2166 (1966).
  3. E.Kay et al.; J.Biol.Chem., 242, 2470 (1967).
  4. R.Lasek et al.; Brain Res., 8, 319 (1968).
  5. W.M.Cowan et al.; Brain Res , 37, 21 (1972).
  6. J.H.La Vail et al.; Brain Res., 58, 470 (1973).
  7. A.M.Graybiel and M.Devor; Brain Res., 68, 167 (1974).
  8. A.H.Bunt et al.; Brain Res., 102, 152 (1976).
  9. D.R.Colman et al.; Brain Res., 102, 156 (1976).
  10. M.Dubois-Dalcq et al.; J.Histochem.Cytochem., 25, 1201 (1977).
  11. M.Sato et al.; Brain Res., 140, 149 (1978).
Table 1. Effect of Various Chemicals on Peroxidase
[The enzyme dissolved in 0.1M phosphate buffer, pH 6.0 (50U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 MIA 2.0 99
Metal salt 2.0   NEM 2.0 97
MgCl₂   102 IAA 2.0 99
CaCl₂   102 Hydroxylamine 2.0 98
Ba(OAc)₂   105 EDTA 5.0 95
FeCl₃   98 o-Phenanthroline 2.0 98
CoCl₂   97 α,α′-Dipyridyl 1.0 96
MnCl₂   97
 Borate 50 98
ZnCl₂   99
 NaF 2.0 98
CdCl₂   99 NaN₃
 2.0 75
NiCl₂   96 Triton X-100 0.10% 98
CuSO₄   98 Brij 35 0.10% 80
Pb(OAc)₂   96 Tween 20 0.10% 89
AgNO₃   91 Span 20 0.10% 98
HgCl₂   92 Na-cholate 0.10% 97
2-Mercaptoethanol
 2.0 94 SDS
 0.05% 98
PCMB 1.0 98 DAC 0.05% 102

※Residual activity was measured by 4AA-DEA method
4AA, 4-Aminoantipyrine; DEA, Diethylaniline
Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

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