TOYOBO Ideas & Chemistry Biotechnology Operating
Department

PYRUVATE OXIDASEE

PREPARATION and SPECIFICATION

Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ　1.5 U/mg-solid or more
Contaminants ATPase ≤ 5.0×10-2 %
GOT, GPT ≤ 5.0×10-2 %
Stabilizers Sugars, FAD

PROPERTIES

Stability Stable at −20 ℃ for at least one year （Fig.1）
Molecular weight approx. 260,000
Isoelectric point 4.3
Michaelis constant 3.4×10-4 M (Pyruvate)
Inhibitors Fe2＋,Zn2＋,Cu2＋,Ag＋,Hg2＋
Optimum pH 5.7 （Fig.2）
Optimum temperature 65 ℃ （Fig.3）
pH Stability pH 5.7−6.5 (25 ℃, 20 hr) （Fig.4）
Thermal stability below 45 ℃ (pH 6.0, 15 min) （Fig.5）
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)

APPLICATIONS

This enzyme is useful for enzymatic determination of pyruvate, GOT and GPT in clinical analysis.

ASSAY

Principle

Principle

The formation of quinoneimine dye is measured at 550 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per minute under the conditions detailed below.

Method

Reagents

A. Pyruvate solution 0.3 M［378 mg of Pyruvate・K salt (MW＝126.15)/10mL of H2O］
B. K-phosphate buffer, pH 5.9 0.15 M
C. 4-Aminoantipyrine solution 0.15 % (150 mg of 4-Aminoantipyrine/100 mL of H2O)
D. EHSPT (TOOS) solution 0.3 %［300 mg of EHSPT (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)/100 mL of H2O］
E. TPP solution 3 mM［13.8 mg of TPP (Thiamine pyrophosphate)(MW＝460.77)/10 mL of H2O］
F. FAD solution 0.15 mM［1.3 mg of FAD・2Na salt (MW＝865.55)/10 mL of H2O］
G. EDTA solution 15 mM［590 mg of EDTA・2Na salt (MW＝394.22)/100 mL of H2O］
H. MgSO4 solution 0.15 M［3.4 g of MgSO4・7H2O(246.48)/100 mL of H2O］
I. Peroxidase solution 50 U/mL［45 mg of peroxidase (110 purpurogallin units/mg)/100 mL of H2O］
J. Enzyme diluent 50 mM K-phosphate buffer, pH 5.7

Procedure

1.Prepare the following working solution in a brownish bottle and store on ice.

10 mL Potassium phosphate buffer, pH 5.9 (B)
2 mL 4-Aminoantipyrine solution (C)
2 mL EHSPT solution (D)
2 mL TPP solutionn (E)
2 mL FAD solution (F)
2 mL EDTA solution (G)
2 mL MgSO4 solution (H)
3 mL Peroxidase (I)
Concentration in assay mixture
Pyruvate 48 mM
Potassium phosphate buffer 50 mM
4-Aminoantipyrine 0.48 mM
EHSPT 0.58 mM
TPP 0.19 mM
FAD 0.01 mM
EDTA 0.97 mM
MgSO4 9.7 mM
Peroxidase Approx. 4.8 U/mL

2.Pipette 2.5 mL of working solution into a cuvette (d ＝ 1.0 cm), add 0.5 mL of pyruvate solution (A), and equilibrate at 37 ℃ for approximately 5 minutes.

3.Add 0.1 mL of the enzyme solution* and mix by gentle inversion.

4.Record the increase in optical density at 550 nm against water for 3 to 4 minutes with a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) using the same method as in the test except that the enzyme diluent is added instead of the enzyme solution.

*Dissolve the enzyme preparation in ice-cold enzyme diluent (J), dilute to 0.1−0.5 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/min (ΔOD test−ΔOD blank)×Vt×df

    36.88×1/2×1.0×Vs

  • ＝ ΔOD/min×1.68×df

Weight activity (U/mg) ＝ (U/mL)×1/C

Vt : Total volume (3.10 mL)
Vs : Sample volume (0.10 mL)
36.88 : Millimolar extinction coefficient of quinoneimine dye under the assay condition (cm2/micromole)
1/2 : Factor based on the fact that one mole of H2O2 produces half a mole of quinoneimine dye.
1.0 : Light path length (cm)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/mL)

REFERENCES

1) L.P.Hager, D.M.Geller and F.Lipman; Fed.Proc.,13, 734 (1954).

2) B.Sedewitz, K.H.Schleifer and F.Gotz; J.Bacteriol,160, 273 (1984).

3) B.Sedewitz, K.H.Schleifer and F.Gotz; J.Bacteriol,160, 462 (1984).

Table 1. Substrate Specificity of Pyruvate oxidase

  • Substrate(50 mM) Relative activity(%)
    Pyruvate 100
    α-Ketobutyrate 5.8
    α-Ketoglutarate 0
    Oxaloacetate 0
    DL-Lactate 0
  • Substrate(50 mM) Relative activity(%)
    Acetate 0
    Acetoacetate 0
    L-Alanine 0
    L-Aspartate 0

Table 2. Effect of Various Chemicals on Pyruvate oxidase

[The enzyme dissolved in 50 mM K-phosphate, pH 6.0 (10 U/mL) was incubated with each chemical at 25 ℃ for 1 hr]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 96
    CaCl2 93
    Ba(OAc)2 97
    FeCl3 8.4
    CoCl2 84
    MnCl2 76
    ZnSO4 48
    Cd(OAc)2 86
    NiCl2 119
    CuSO4 0.9
    Pb(OAc)2 33
    AgNO3 0
    HgCl2 0
    PCMB 1.0 66
    MIA 2.0 96
  • Chemical Concn.(mM) Residual
    activity(%)
    NaF 2.0 100
    NaN3 20 94
    EDTA 5.0 107
    o-Phenanthroline 2.0 97
    α,α′-Dipyridyl 1.0 95
    Borate 50 102
    IAA 2.0 102
    NEM 2.0 104
    Hydroxylamin 2.0 98
    Triton X-100 0.10 % 143
    Brij 35 0.10 % 133
    Tween 20 0.10 % 146
    Span 20 0.10 % 121
    Na-cholate 0.10 % 116
    SDS 0.05 % 85
    DAC 0.05 % 53

Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate;
IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylallkylammonium chloride.

  • Fig.1. Stability (Power form)

    Fig.1. Stability (Power form)

    (kept under dry conditions)

  • Fig.2. pH-Activity

    Fig.2. pH-Activity

    (37 ℃ in 50 mM K-phosphate buffer)

  • Fig.3. Temperature activity

    Fig.3. Temperature activity

    (in 50 mM K-phosphate buffer, pH 5.7)

  • Fig.4. pH-Stability

    Fig.4. pH-Stability

    25 ℃, 20 hr-treatment with 50 mM buffer solution (contg. 10 mM MgSO4. 10 μM FAD, 0.2 mM TPP):pH 4.0-6.0, acetate;pH 5.7-8.0 K-phosphate; pH 7.5-9.0,Tris-HCI

  • Fig.5. Thermal stability

    Fig.5. Thermal stability

    15 min-treatment with 50 mM K-phosphate buffer(cong. 10 mM MgSO4. 10 μM FAD, 0.2 mM TPP), pH 6.0. enzyme concn.:10 U/mL

活性測定法（Japanese）

1. 原理

原理

4-AminoantipyrineとEHSPTの酸化縮合生成物である Quinoneimine色素を550nmで測定し,上記反応で生成したH2O2量を定量する。

2.定義

下記条件で1分間に1マイクロモルのH2O2を生成する酵素量を1単位(U)とする。

3.試薬

  • 0.3 Mピルビン酸水溶液〔378 mgのピルビン酸・K塩 (MW＝126.15)を10 mLの蒸留水に溶解する。〕
  • 0.15 Mリン酸カリウム緩衝液，pH 5.9
  • 0.15 % 4-AA水溶液(150 mgの4-アミノアンチピリン を100 mLの蒸留水に溶解する。)
  • 0.3 % EHSPT(TOOS)水溶液〔300 mgのEHSPT を100 mLの蒸留水に溶解する〕
  • 3.0 mM TPP水溶液〔13.8 mgのTPP(MW＝ 460.77)を10 mLの蒸留水に溶解する。〕
  • 0.15 mM FAD水溶液〔1.3 mgのFAD･2Na塩 (MW＝865.55)を10 mLの蒸留水に溶解する。〕
  • 15 mM EDTA水溶液〔590 mgのEDTA･2Na塩 (MW＝394.22)を100 mLの蒸留水に溶解する。〕
  • 0.15 M MgSO4水溶液〔3.4 gのMgSO4･7H2O (MW＝246.48)を100 mLの蒸留水に溶解する。〕
  • 50 U/ml POD水溶液〔45 mgペルオキシダーゼ (POD)(110プルプロガリン単位/mg)を100 mLの蒸留水に溶解する。〕

酵素溶液：酵素標品を予め氷冷した50 mM K-リン酸緩衝液，pH 5.7で溶解し,分析直前に同緩衝液で0.1〜0.5 U/mLに希釈する。

4.手順

1.下記反応混液を調製する(褐色瓶にて氷冷保存)。

10 mL K-リン酸緩衝液 (B)
2 mL 4-AA水溶液 (C)
2 mL EHSPT水溶液 (D)
2 mL TPP水溶液 (E)
2 mL FAD水溶液 (F)
2 mL EDTA水溶液 (G)
2 mL MgSO4水溶液 (H)
3 mL POD水溶液 (I)

2.反応混液2.5 mLをキュベット(d ＝ 1.0cm)に採り,ピルビン酸水溶液(A)0.5 mLを添加し,37 ℃で約5分間予備加温する。

3.酵素溶液0.1 mLを添加し,ゆるやかに混和後,水を対照に37 ℃に制御された分光光度計で550 nmの吸光度 変化を3～4分間記録し,その初期直線部分から1分間 当りの吸光度変化を求める(ΔODtest)。

4.盲検は反応混液①に酵素溶液の代わりに酵素希釈液(50mM K-リン酸緩衝液,pH 5.7)を0.1ml加え,上記同様に操作を行って,1分間当たりの吸光度変化を求める(ΔODblank)。

5.計算式

  • U/mL =

  • ΔOD/min (ΔOD test−ΔOD blank)×3.1(mL)×希釈倍率

    36.88×1/2×1.0×0.1(mL)

＝ ΔOD/min×1.68×希釈倍率
U/mg = U/mL×1/C
36.88 : Quinoneimine色素の上記測定条件下でのミリモル分子吸光係数(cm2/micromole)
1/2 : 酵素反応で生成したH2O2の1分子から形成するQuinoneimine色素は1/2分子である事による係数。
1.0 : 光路長(cm)
C : 溶解時の酵素濃度(c mg/mL)