PEROXIDASE

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℃ for at least one year (Fig.1,2,3)
Molecular weight approx. 40,000
Structure Glycoprotein with one mole of protohaeminIX 2)
Inhibitors Cyanide, sulfide, fluoride, azide 3
Optimum pH 6.0−7.0 (Fig.6)
Optimum temperature 45℃ (Fig.7)
pH Stability pH 5.0−10.0 (25℃, 20hr) (Fig.8)
Thermal stability below 50℃ (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

Principle

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

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

14.0ml H2O
2.0ml Pyrogallol solution (A)
1.0ml H2O2 solution (B)
2.0ml Phosphate buffer, pH6.0 (C)
Concentration in assay mixture
Phosphate buffer 15 mM
Pyrogallol 40 mM
H2O2 7.4mM

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

3.After exactly 20 seconds at 20℃, 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℃, 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 :

  • Volume activity (U/ml) =

  • ΔOD (OD test−OD blank)×df


    0.117×Vs

  • = ΔOD×8.547×df

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℃.

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℃ for 1hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 102
    CaCl2 102
    Ba(OAc)2 105
    FeCl3 98
    CoCl2 97
    MnCl2 97
    ZnCl2 99
    CdCl2 99
    NiCl2 96
    CuSO4 98
    Pb(OAc)2 96
    AgNO3 91
    HgCl2 92
    2-Mercaptoethanol 2.0 94
    PCMB 1.0 98
  • Chemical Concn.(mM) Residual
    activity(%)
    MIA 2.0 99
    NEM 2.0 97
    IAA 2.0 99
    Hydroxylamine 2.0 98
    EDTA 5.0 95
    o-Phenanthroline 2.0 98
    α,α′-Dipyridyl 1.0 96
    Borate 50 98
    NaF 2.0 98
    NaN3 2.0 75
    Triton X-100 0.10% 98
    Brij 35 0.10% 80
    Tween 20 0.10% 89
    Span 20 0.10% 98
    Na-cholate 0.10% 97
    SDS 0.05% 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.

  • Fig.1. Stability (PEO-131) (Powder form)

    Fig.1. Stability (PEO-131)
    (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (PEO-301) (Powder form)

    Fig.2. Stability (PEO-301)
    (Powder form)

    (kept under dry conditions)

  • Fig.3. Stability (PEO-302) (Powder form)

    Fig.3. Stability (PEO-302)
    (Powder form)

    (kept under dry conditions)

  • Fig.4. Stability (Powder form)

    Fig.4. Stability (Powder form)

    (kept under dry conditions)

  • Fig.5. Stability (Powder form)

    Fig.5. Stability (Powder form)

    (kept under dry conditions)

  • Fig.6. pH-Activity

    Fig.6. pH-Activity

    20℃, 20sec-reaction in 0.1M buffer solution: pH4.0-6.0, acetate; pH6.0-8.0, phosphate

  • Fig.7. Temperature activity

    Fig.7. Temperature activity

    20sec-reaction in 0.1M phosphate buffer, pH6.0

  • Fig.8. pH-Stability

    Fig.8. pH-Stability

    25℃, 20hr-treatment with 50mM buffer solution: pH3.5-6.0, acetate; pH6.0-8.0, phosphate; pH9.0-11.0 borate

  • Fig.9. Thermal stability

    Fig.9. Thermal stability

    10min-treatment with 50mM phosphate buffer, pH6.0

活性測定法(Japanese)

1. 原理

原理

生成するPurpurogallinをエーテル抽出し,420nmの吸光度の変化で測定する。

2.定義

下記条件下で20秒間に1.0mgのPurpurogallinを生成する酵素量を1Purpurogallin単位(U)とする。

3.試薬

  • 5%(W/V)ピロガロール水溶液(用時調製)
  • 0.147M H2O2水溶液〔30%(W/V)H2O2溶液 1.67mlを蒸留水で希釈して100mlとする〕 (用時調製)
  • 0.1Mリン酸緩衝液,pH6.0(反応混液及び酵素希釈用)
  • 2.0N H2SO4溶液

酵素溶液:酵素標品を予め氷冷した0.1Mリン酸緩衝液, pH6.0で溶解し,同緩衝液で3.0〜 6.0Purpurogallin U/mlに希釈して氷冷保存する。

4.手順

1.試験管(32φ×200mm)に下記反応混液を調製し, 20℃で約5分間予備加温する。

14.0ml 蒸留水
2.0ml ピロガロール水溶液 (A)
1.0ml H2O2水溶液 (B)
2.0ml リン酸緩衝液 (C)

2.酵素溶液1.0mlを加え,反応を開始する。

3.20℃で正確に20秒間反応させた後, H2SO4溶液 (D)1.0mlを加えて反応を停止させる。反応停止後の混液から生成したPurpurogallinをエーテル15mlで抽出する。この操作を5回繰り返し,抽出液を合わせ,更にエーテルを加えて全量を100mlにする。この液につき 420nmにおける吸光度を測定する(OD test)。

4.盲検は反応混液①を20℃で20秒間放置後, H2SO4 溶液(D)1.0mlを加えて混和し,次いで酵素溶液1.0mlを加えて調製する。この液につき上記同様にエーテル抽出を行って吸光度を測定する(ODblank)。

5.計算式

  • U/ml =

  • ΔOD (OD test−OD blank)×希釈倍率


    0.117×1(ml)

= ΔOD×8.547×希釈倍率
U/mg = U/ml×1/C
0.117 : 1mg% Purpurogallinエーテル溶液の420nmにおける吸光度
C : 溶解時の酵素濃度(c mg/ml)
(注)1Purpurogallin単位は13.5国際単位(o-dianisidine を基質とし, 25℃の反応条件下)に相当する。