TOYOBO Ideas & Chemistry バイオ事業総括部

HBH-311

PREPARATION and SPECIFICATION

Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ　20U/mg-solid or more
(containing approx. 40% of stabilizers)
Contaminant NADPH oxidase ≤1.0×10-1%
Stabilizers Sugars, FAD

PROPERTIES

Stability Stable at −20℃ for at least one year(Fig.1)
Molecular weight 55,000〜60,000
Michaelis constants 2.0×10-5M (p-Hydroxybenzoate), 4.0×10-5M (NADPH)
Structure One mol of FAD per mol of enzyme
Inhibitors Ag＋, Hg＋＋, PCMB, SDS
Optimum pH 7.7−7.9(Fig.3)
Optimum temperature 35℃(Fig.4)
pH Stability pH 5.0−7.5 (25℃, 72hr)(Fig.5)
Thermal stability below 40℃ (pH 6.0, 15min)(Fig.6)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)

APPLICATIONS

This enzyme is useful for enzymatic determination of choline esterase when coupled with protocatechuate 3, 4-dioxygenase (PCO-302)

ASSAY

Principle

Principle

The disappearance of NADPH is measured at 340nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of NADPH per minute under the conditions described below.

Method

Reagents

A. Tris-malate buffer, pH 8.2 50mM［Dissolve 3.03g of Tris (M.W＝121.14) in ca.300ml of H2O and, after adjusting the pH to 8.2 at 25℃ with 1.0M maleic acid, fill up to 500ml with H2O.］
B. p-hydroxybenzoate solution 5.0mM［80mg p-hydroxybenzoate (Na salt)/100ml of buffer solution (A)］(Should be prepared fresh)
C. FAD solution 0.2mM［19mg FAD・Na2 /100ml or buffer solution (A)］(Should be prepared fresh)
D. NADPH solution 3.0mM［272mg NADPH・Na4・4H2O/100ml of buffer solution (A)］(Should be prepared fresh)
E. Enzyme diluent 50mM K-phosphate buffer, pH 6.0 containing 0.2% BSA

Procedure

1. Prepare the following working solution (10 tests) in a brownish bottle and store on ice.

21.0ml Buffer solution (A)
3.0ml Substrate solution (B)
3.0ml FAD solution (C)
3.0ml NADPH solution (D)
Concentration in assay mixture
Tris-malate buffer 49 mM
p-Hydroxybenzoate 0.49mM
FAD 20 μM
NADPH 0.30mM

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

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

4. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37℃ and calculate the ΔOD per minute from 1.5 to 3 minutes portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (E) is added instead of enzyme solution.

*Dissolve the enzyme preparation in ice-cold enzyme diluent (E) (1.0mg/ml or more) and dilute to 0.2−0.6 U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

  • Volume activity (U/ml) =

  • ΔOD/min (ΔOD test−ΔOD blank)×Vt×df

    6.22×1.0×Vs

  • ＝ ΔOD/min×9.8×df

Weight activity (U/mg) ＝ (U/ml)×1/C

Vt : Total volume (3.05ml)
Vs : Sample volume (0.05ml)
6.22 : Millimolar extinction coefficient of NADPH (cm2/micromole)
1.0 : Light path length (cm)
df : Dilution factor
C : Enzyme concentration (c mg/ml)

REFERENCES

1) H.Shoun and K.Arima; Protein, Nucleic acid and Enzyme, 25, 820, (1980).

2) K.Yano and K.Arima; Agric.Biol.Chem., 33, 689 (1969).

3) K.Hosokawa and R.Y.Stanier; J.Biol.Chem., 241, 2453 (1966).

Table 1. Substrate Specificity of p-Hydroxybenzoate hydroxylase

  • Substrate(0.5mM) Relative activity(%)
    p-Hydroxybenzoic acid 100
    Methyl-p-hydroxybenzoic acid <0.05
    Ethyl-p-hydroxybenzoic acid <0.05
    n-Propyl-p-hydroxybenzoic acid <0.05
    m-Hydroxybenzoic acid <0.05
    o-Hydroxybenzoic acid
  • Substrate Relative activity(%)
    Protocatechuic acid 3.3
    β-Resorcylic acid 4.5
    Gentisic acid <0.05
    p-Chlorobenzoic acid <0.05
    p-Aminobenzoic acid 0.12

Table 2. Effect of Various Chemicals on p-Hydroxybenzoate hydroxylase (Residual activity after 1 hr-treatment at 30℃)

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 1.0
    CoCl2 106
    ZnCl2 94
    CuSO4 103
    AgNO3 0
    MgSO4 107
    BaCl2 107
    FeCl3 106
    MnCl2 90
    NiCl2 104
    CaCl2 97
    SnCl2 102
    HgCl2 1.1
    CrCl2 93
    CdCl2 104
    FeSO4 90
  • Chemical Concn.(mM) Residual
    activity(%)
    MIA 1.0 91
    PCMB 1.0 3.7
    NaN3 1.0 97
    NaF 1.0 96
    o-Phenanthroline 1.0 95
    α,α′-Dipyridyl 1.0 90
    EDTA 5.0 96
    Borate 50 104
    Tween 20 0.1% 91
    Brij 35 0.1% 101
    Span 20 0.1% 94
    Triton X-100 0.1% 97
    Na-cholate 0.1% 88
    SDS 0.05% 34

MIA, Monoiodoacetate; PCMB, p-Chloromercuribenzoate; EDTA, Ethylenediaminetetraacetate; SDS, Sodium dodecyl sulfate.

  • Fig.1. Stability (Powder form)

    Fig.1. Stability (Powder form)

    (kept under dry conditions)

  • Fig.2. Stability (Liquid form at 25℃)

    Fig.2. Stability (Liquid form at 25℃)

    enzyme concentration:500U/ml buffer compostion:50mM K-phosphate buffer,pH6.0

  • Fig.3. pH-Activity

    Fig.3. pH-Activity

    37℃ in 50mM buffer solution: ○̶̶○,K-phosphate; △̶̶△,Trismalate

  • Fig.4. Temperature activity

    Fig.4. Temperature activity

    (in 50mM Tris-malate buffer, pH8.2)

  • Fig.5. pH-Stability

    Fig.5. pH-Stability

    25℃ in 72hr-treatment with 50mM buffer solution:△̶̶△,acetate; ○̶̶○,K-phosphate;×̶̶×,glycine-NaOH

  • Fig.6. Thermal stability

    Fig.6. Thermal stability

    15min-treatment with 50mM K-phosphate buffer,pH6.0

活性測定法（Japanese）

1. 原理

原理

NADPHの消失量を340nmの吸光度の変化で測定する｡

2.定義

下記条件下で1分間に1マイクロモルのNADPHが酸化される酵素量を1単位(U)とする。

3.試薬

  • 50mM Tris-malate緩衝液pH8.2(3.03gのTris(MW＝121.14)を約300mlの蒸留水で溶解し,1.0M マレイン酸でpHを8.2(25℃)に調整後,蒸留水で500mlにする)
  • 5.0mM p-ヒドロキシ安息香酸溶液(80mgのp-ヒドロキシ安息香酸ナトリウムを100mlの緩衝液(A)で溶解する)(用時調製)
  • 0.2mM FAD溶液(19mgのFAD・Na2を約100mlの緩衝液(A)で溶解する)(用時調製)
  • 3.0mM NADPH溶液(272mgのNADPH・Na4-4H2Oを約100mlの緩衝液(A)で溶解する)(用時調製)

酵素溶液：酵素標品を予め氷冷した0.2% BSAを含む50mM K-リン酸緩衝液pH6.0で溶解(1.0mg/ml以上)し，分析直前に同緩衝液で0.2〜0.6U/mlに希釈する。

4.手順

1.下記反応混液を調製する(用時調製し,褐色瓶で氷冷保存)。

21.0ml Tris-malate緩衝液 (A)
3.0ml 基質溶液 (B)
3.0ml FAD溶液 (C)
3.0ml NADPH溶液 (D)

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

3.酵素溶液0.05ml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340nmの吸光度変化を1.5〜3.0分間記録し,その1.5〜3分間の吸光度から1分間当りの吸光度変化を求める(ΔODtest)。

4.盲検は反応混液に酵素溶液の代りに酵素希釈液(0.2% BSAを含む50mM K-リン酸緩衝液pH6.0)を0.05ml加え,上記同様に操作を行って,1分間当りの吸光度変化を求める(ΔOD blank)。

5.計算式

  • U/ml =

  • ΔOD/min (ΔOD test−ΔOD blank)×3.05(ml)×希釈倍率

    6.22×1.0×0.05(ml)

＝ ΔOD/min×9.8×希釈倍率
U/mg = U/ml×1/C
6.22 : NADPHのミリモル分子吸光係数(cm2/micromole)
1.0 : 光路長(cm)
C : 溶解時の酵素濃度(c mg/ml)