TOYOBO Ideas & Chemistry Biotechnology Operating
Department

PREPARATION and SPECIFICATION

Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ　10 U/mg-solid or more
　　　　　　(containing approx. 20 % of stabilizers)
Contaminant Catalase ≤1.0×102 %
Stabilizers EDTA, BSA, amino acids (glycine, sodium glutamate, etc.)

PROPERTIES

StabilityStable at −20 ℃ for at least one year （Fig.1）
Molecular weightapprox. 95,000
Isoelectric point4.1±0.1
Michaelis constants2.84×10-3 M (Choline), 5.33×10-3 M (Betaine aldehyde)
StructureOne mol of FAD is covalently bound to mol of the enzyme 8)
Inhibitors p-Chloromercuribenzoate, Cu2+,Co2+,Hg2+,Ag＋
Optimum pH8.0−8.5（Fig.4）
Optimum temperature40−45 ℃（Fig.5）
pH StabilitypH 7.0−9.0 (30 ℃, 2 hr)（Fig.6）
Thermal stabilitybelow 37℃ (pH 7.5, 10 min)（Fig.7）
Effect of various chemicals(Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of phospholipids in combination with phospholipase D and for measuring choline esterase activity in clinical analysis.9〜11)

ASSAY

Principle

Principle

The formation of quinoneimine dye is measured at 500 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. Choline chloride solution 2.1 %［2.1g of choline chloride/100 mL of Tris-HCl buffer (D)］(Should be prepared fresh)
B. 4-AA solution 1.0 % (1.0 g of 4-aminoantipyrine/100 mL of H2O)(Store at 4 ℃ in a brownish bottle)
C. Phenol solution 1.0 % (1.0 g of phenol/100 mL of H2O; store at 4 ℃ in a brownish bottle)
D. Tris-HCl buffer 0.1 M Tris-HCl buffer, pH 8.0［Dissolve 12.1 g of Tris (MW＝121.14) in approx. 800 mL of H2O and, after adjusting the pH to 8.0 at 25℃ with 2.0 N HCl, make up to 1,000 mL with H2O.］
E. Enzyme diluent 10 mM Tris-HCl buffer, pH 8.0, containing 2mM EDTA and 1.0 % KCl.

Procedure

1.Prepare the following working solution (100 mL) in a brownish bottle before use and store on ice.

97 mL Substrate solution (A)
1.0 mL 4-AA solution (B)
2.0 mL Phenol solution (C)
5.0 mL Peroxidase from horseradish
(110 purpurogallin units/mg)
(Toyobo GradeⅢ)
Concentration in assay mixture
Tris buffer 97 mM
Choline chloride 0.14 M
EDTA 33 μM
KCI 2.2 mM
4-Aminoantipyrine 0.48 mM
Phenol 2.1 mM
POD approx. 4.92 U/mL

2.Pipette 3.0 mL of working solution into a cuvette (d ＝ 1.0 cm) and equilibrate at 37 ℃ for approx. 5 minutes.

3.Add 0.05 mL of the enzyme solution* and mix by gentle inversion.

4.Record the increase in optical density at 500 nm against the working solution for 3 to 4 minutes in a spectrophotometer thermostated at 37 ℃, and calculate the ΔOD per minute from the initial linear portion of the curve.

*Dissolve the enzyme preparation in ice-cold Tris-HCl buffer (D) and dilute to 0.1−0.5 U/mL with enzyme diluent (E).

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/mL) =

  • ΔOD/min×Vt×df

    12.0×1/2×1.0×Vs

＝ ΔOD/min×10.17×df

Weight activity (U/mg) ＝ (U/mL)×1/C

Vt : Total volume (3.05 mL)
Vs : Sample volume (0.05 mL)
12.0 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions(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)P.J.G Mann and J.H.Quastel; Biochem.J.,31, 869 (1937).

2)P.J.G Mann et al; Biochem.J.,32, 1024 (1938).

3)H.S.Shieh; Can.J.Microbiol., 10, 837 (1964).

4)H.S.Shieh; Ibid.,11, 375 (1965).

5)G.J.J.Kortstee; Arch.Mikirobiol., 71, 235 (1970).

6)S.Ikuta, K.Matuura, S.Imamura, H.Misaki, Y.Horiuti; J.Biochem., 82, 157 (1977).

7)S.Ikuta, S.Imamura, H.Misaki and Y.Horiuti ; J.Biochem., 82, 1741 (1977).

8)M.Ohta-Fukuyama, Y.Miyake, S.Emi and T.Yamano; J.Biochem., 88, 197 (1980).

9)M.Takayama et al; Clin. Chim. Acta, 79, 93 (1977).

10)K.Sugawara and A.Kihara; Eisei Kensa, 27(1), 106 (1978).

11)H.Okabe et al ; Clin.Chim.Acta, 80, 87 (1977).

Table 1. Effect of Various Chemicals on Choline oxidase

［The enzyme dissolved in 10mM Tris-HCl buffer, pH 8.0 contg. 2mM EDTA and 1.0 % KCl (5U/mL) was incubated with each chemical at 25 ℃ for 1hr.］

  • Chemical Concn.(mM) Residual activity(%)
    None ー 100
    Metal salt 2.0
    MgCl2 87
    CaCl2 92
    Ba(OAc)2 89
    FeCl3 87
    CoCl2 89
    MnCl2 91
    ZnCl2 88
    CdCl2 92
    NiCl2 91
    CuSO4 92
    Pb(OAc)2 87
    AgNO3 80
    HgCl2 48
    2-Mercaptoethanol 2.0 90
    PCMB 1.0 13
  • Chemical Concn.(mM) Residual activity(%)
    MIA 2.0 87
    NEM 2.0 100
    IAA 2.0 95
    Hydroxylamine 2.0 77
    EDTA 5.0 92
    o-Phenanthroline 2.0 90
    α,α′-Dipyridyl 1.0 91
    Borate 50 94
    NaF 2.0 92
    NaN3 2.0 92
    Triton X-100 0.10 % 96
    Brij 35 0.10 % 92
    Tween 20 0.10 % 95
    Span 20 0.10 % 94
    Na-cholate 0.10 % 96
    SDS 0.05 % 95
    DAC 0.05 % 91
Ac, CH3CO;PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; NEM, N-Ethylmaleimide; IAA, Iodoacetamide;EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (Powder form)

    Fig.2. Stability (Powder form)

    (kept under dry conditions)

  • Fig.3. Stability (Liquid form at 37℃)

    Fig.3. Stability (Liquid form at 37 ℃)

    enzyme concentration: 1.0mg/mL buffer composition: 0.1M K-phosphate buffer, pH7.5

  • Fig.4. pH-Activity

    Fig.4. pH-Activity

    (37 ℃, in 50mM K-phosphate buffer)

  • Fig.5. Temperature activity

    Fig.5. Temperature activity

    (in 50mM K-phosphate buffer,pH7.5)

  • Fig.6. pH-Stability

    Fig.6. pH-Stability

    30 ℃, 2hr-treatment with 50mM buffer solution:pH6.0-9.0, K-phosphate; pH9.0-10.0, glycine-NaCl-NaOH.

  • Fig.7. Thermal stability

    Fig.7. Thermal stability

    15min-treatment with 50mM K-phosphate buffer, pH7.5

活性測定法（Japanese）

1. 原理

原理

4-AminoantipyrineとPhenolの酸化縮合生成物であるQuinoneimine色素を500nmで測定し,上記反応で生成したH2O2量を定量する。

2.定義

下記条件下で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

  • 2.1 %塩化コリン溶液(2.1gの塩化コリンを0.1MTris-HCl緩衝液,pH8.0で溶解し100 mLとする)
  • 1.0 % 4-AA水溶液(1.0gの4-アミノアンチピリンを蒸留水に溶解して100 mLとする)(褐色瓶中で4 ℃保存)
  • 1.0 %フェノール水溶液(1.0gのフェノールを蒸留水に溶解して100 mLとする)(褐色瓶中で4 ℃保存)
  • 0.1M Tris-HCl緩衝液，pH8.0〔12.1gのトリス(MW＝121.14)を約800 mLの蒸留水で溶解し,2.0NHClでpH8.0(25 ℃)に調製した後1000 mLにする〕

酵素溶液：酵素標品を予め氷冷した0.1M Tris-HCl緩衝液,pH8.0で溶解し,2.0mM EDTAと1.0 %のKClを含む10mM Tris-HCl緩衝液,pH8.0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)。

97.0 mL 基質溶液 (A)
1.0 mL 4-AA水溶液液 (B)
2.0 mL フェノール水溶液 (C)
5.0 mL peroxidase(110プルプロガリン単位/mg)

2.反応混液3.0 mLをキュベット(d ＝ 1.0 cm)にとり,37℃で約5分間予備加温する。

3.酵素溶液0.05 mLを添加し，ゆるやかに混和し,反応混液を対照に37℃に制御された分光光度計で500nm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分間あたりの吸光度変化を求める(ΔOD/min)。

5.計算式

  • U/mL =

  • ΔOD/min×3.05(mL)

    12.0×1/2×1.0×0.05(mL)

  • ×希釈倍率

＝ ΔOD/min×10.17×希釈倍率
U/mg ＝U/mL×1/C
12.0 : 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 : 酸素反応で生成したH2O2の1分子のから形成する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)