TOYOBO Ideas & Chemistry Biotechnology Operating
Department

HBD-301

PREPARATION and SPECIFICATION

Appearance White amorphous powder, lyophilized
Activity GradeⅢ　100 U/mg-solid or more
Contaminants Malate dehydrogenase ≤ 2.0×10-3 %
Lactate dehydrogenase ≤ 2.0×10-3 %
NADH oxidase ≤ 2.0×10-3 %
Stabilizers Sucrose, mannitol, BSA

PROPERTIES

Stability Stable at −20 ℃ for at least one year(Fig.1)
Molecular weight approx. 130,000 (by gel filtration)
Isoelectric point 5.6±0.1
Michaelis constants 4.2×10-4 M (25 ℃, pH 8.3), 7.0×10-4 M (37 ℃, pH 8.3) ( D-3-Hydroxybutyrate)
4.9×10-5 M (25 ℃, pH 8.3), 7.2×10-5 M (37 ℃, pH 8.3)(NAD＋)
8.1×10-5 M (25 ℃, pH 7.1), 2.4×10-4 M (37 ℃, pH 7.1)(Acetoacetate)
8.4×10-6 M (25 ℃, pH 7.1), 1.5×10-5 M (37 ℃, pH 7.1)(NADH)
Inhibitors PCMB, MIA, IAA, Ag＋, Hg2＋, SDS, DAC
Optimum pH 8.3(Fig.3)
Optimum temperature 55 ℃(Fig.4)
pH Stability pH 5.0−8.5 (25 ℃, 20 hr)(Fig.5)
Thermal stability below 40 ℃ (pH 6.5, 15 min)(Fig.6)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)

APPLICATIONS

This enzyme is useful for enzymatic determination of ketone bodies (D-3-hydroxybutyrate and acetoacetate) in clinical analysis.

ASSAY

Principle

Principle

The formation of NADH is measured at 340 nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of NADH per minute under the conditions detailed below.

Method

Reagents

A. Tris-HCl buffer, pH 8.5 (25 ℃) 0.1 M
B. 3-Hydroxybutyrate solution 158 mM［200 mg D,L-3-Hydroxybutyrate Na salt (MW＝126.09)/10 mL of Tris-HCl buffer (A)］(Stable at least 5 days if stored at 4 ℃)
C. NAD＋ solution 27.9 mM［80 mg NAD＋・3H2O (MW＝717.45)/4.0 mL of Tris-HCl buffer (A)］(Stable for at least 5 days if stored at 4 ℃)
D. Enzyme diluent 0.1 M Tris-HCl buffer, pH 8.5 contg. 0.1 % BSA

Procedure

1. Prepare the following reaction mixture in a cuvette (d ＝ 1.0 cm) and equilibrate at 37 ℃ for approximately 5 minutes.

2.3 mL Tris-HCl buffer, pH 8.5 (A)
0.5 mL Substrate solution (B)
0.2 mL NAD＋ solution (C)
Concentration in assay mixture
Tris-HCl buffer 0.1 M
3-Hydroxybutyrate 25 mM
NAD＋ 1.8 mM

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

3. Record the increase in optical density at 340 nm against water for 2 to 3 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 the test except that the enzyme diluent is added instead of enzyme solution.

*Dissolve the enzyme preparation in ice-cold enzyme diluent (D), 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

    6.22×1.0×Vs

  • ＝ ΔOD/min×4.98×df

Weight activity (U/mg) ＝ (U/mL)×1/C

Vt : Total volume (3.1 mL)
Vs : Sample volume (0.1 mL)
6.22 : Millimolar extinction coefficient of NADH at 340 nm (㎠/micromole)
1.0 : Light path length (cm)
df : Dilution factor
C : Enzyme concentration in dissolution (c mg/mL)

REFERENCES

1) H.U.Bergmeyer, K.Gawehn, H.Klotzsh, H.A.Krebs and D.H.Williamson; Biochem.J., 102, 423 (1967).

2) F.P.Delafield, K.E.Cooksey and M.Doudoroff; J.Biol.Chem., 240, 4023 (1965).

3) C.W.Shuster and M.Doudoroff; J.Biol.Chem., 237, 603 (1962).

4) I.Sekuzu, P.Jurtshuk and D.E.Green; J.Biol.Chem., 238, 975 (1963).

5) J.D.Smiley and G.Ashwell; J.Biol.Chem., 236, 357 (1961).

Table 1. Substrate Specificity of D-3-Hydroxybutyrate dehydrogenase

  • Substrate Relative activity(%)
    3-Hydroxybutyrate 100
    3-Hydroxypropionate 0.14
    Lactate 0
    Glycerate 0
    2-Hydroxybutyrate 0
    L-Malate 0
    D,L-Malate 0
  • Substrate Relative activity(%)
    sec-Butyl alcohol 0
    Gluconate 0
    Glycolate 0.04
    NAD+ 100
    NADP+ 4.74

Table 2. Effect of Various Chemicals on D-3-Hydroxybutyrate dehydrogenase

[The enzyme dissolved in 50 mM K-phosphate buffer, pH 6.5(10 U/mL) was incubated at 25℃ for 1hr.]

  • Chemical Concn.(mM) Residual
    activity(%)
    None - 100
    Metal salt 2.0
    MgCl2 105
    CaCl2 101
    Ba(OAc)2 98
    FeCl3 101
    CoCl2 102
    MnCl2 103
    ZnSO4 100
    Cd(OAc)2 100
    NiCl2 103
    CuSO4 83
    Pb(OAc)2 96
    AgNO3 2.5
    HgCl2 0
    PCMB 2.0 0
    MIA 2.0 1
  • Chemical Concn.(mM) Residual
    activity(%)
    NaF 2.0 100
    NaN3 20 104
    EDTA 5.0 97
    o-Phenanthroline 2.0 96
    α,α′-Dipyridyl 1.0 97
    Borate 50 103
    IAA 2.0 4
    NEM 2.0 59
    Hydroxylamine 2.0 101
    Triton X-100 0.10 % 113
    Brij 35 0.10 % 37
    Tween 20 0.10 % 68
    Span 20 0.10 % 104
    Na-cholate 0.10 % 107
    SDS 0.05 % 5
    DAC 0.05 % 4

Ac, CH3CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, lodoacetamide; NEM, N-Ethylmaleimide; 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. pH-Activity

    Fig.3. pH-Activity

    37 ℃,5 min-reaction in 0.1 M buffer solution: pH 5.3-6.5, dimethylglutaric acid-NaOH;pH 5.9-8.3,K-phosphate;pH 7.9-9.1,Tris-HCI: pH 8.9-9.2,K2CO3-NaHCO3

  • Fig.4. Temperature activity

    Fig.4. Temperature activity

    (in 0.1 M Tris-HCI buffer, pH 8.3)

  • Fig.5.pH-Stability

    Fig.5.pH-Stability

    25 ℃,20 hr-treatment with 50 mM buffer solution:pH 4.0-6.0, dimethylglutaric acidNaOH;pH 6.0-8.0, K-phosphate;pH 8.0-9.0, Tris-HCI;pH 9.0-10.5, K2CO3-NaHCO3

  • Fig.6. Thermal stability

    Fig.6. Thermal stability

    15 min-treatment with 50 mM K-phosphate buffer,pH 6.5. enzyme concn.:50 U/mL

活性測定法（Japanese）

1. 原理

原理

NADHの生成量を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FロモルのNADHを生成する酵素量を1単位(U)とする。

3.試薬

  • 0.1M Tris-HCl緩衝液，pH 8.5 (25℃)
  • 158mM 3-ヒドロキシ酪酸溶液(200mgのD,L-3-ヒドロキシ酪酸ナトリウム塩 (MW＝126.09)を10 mLのTris-HCl緩衝液(A)に溶解する)(4℃保存で，少なくとも５日間は使用可能)
  • 27.9mM NAD＋溶液(80mgのNAD＋・3H2O(MW＝717.45)を4.0 mLのTris-HCl緩衝液(A)に溶解する)(4℃保存で，少なくとも５日間は使用可能)

酵素溶液：酵素標品を予め氷冷した0.1 %牛血清アルブミンを含む0.1M Tris-HCl緩衝液,pH8.5で溶解し，同緩衝液で0.1〜0.5 U/ mLに希釈して氷冷保存する。

4.手順

1.下記反応混液をキュベット(d＝1.0cm)に調製し,37℃で約5分間予備加温する。

2.3 mL 0.1MTris-HCl緩衝液 (A)
0.5 mL 基質溶液 (B)
0.2 mL NAD＋溶液 (C)

2.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340nmの吸光度変化を2〜3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(ΔOD test)。

3.盲検は反応混液①に酵素溶液の代りに酵素希釈液(0.1 %牛血清アルブミンを含む0.1MTris-HCl緩衝液,pH 8.5)を0.1 mL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)×3.1(mL)×希釈倍率

    6.22×1.0×0.1(mL)

＝ ΔOD/min×4.98×希釈倍率
U/mg = U/mL×1/C
6.22 : NADH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)