D-3-HYDROXYBUTYRATE DEHYDROGENASE from Achromobacter sp.

HBD-311

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅢ 450U/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, Synthetic polymer
PROPERTIES
Stability:  Please contact us.(Fig.1)
Molecular weight :  approx.  28,000 (by gel filtration)
Isoelectric point :  5.6±0.1
Michaelis constants :  1.9×10⁻³M (37°C, pH8.3) (D-3-Hydroxybutyrate)
1.6×10⁻⁴M (37°C, pH8.3)(NAD⁺)
Inhibitors :  Fe3＋, NEM
Optimum pH :  8.0-8.5(Fig.2)
Optimum temperature :  50°C(Fig.3)
pH Stability :  pH 5.5-10 (25°C, 20hr)(Fig.4)
Thermal stability :  below 40°C (pH 6.5, 15min)(Fig.5)
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:

D-3-hydroxybutyrate dehydrogenase

D-3-Hydroxybutyrate+NAD⁺                                                  ► Acetoacetate+NADH+H⁺


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

Unit definition:

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

Method:

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

Procedure

Concentration in assay mixture
Tris-HCl buffer 0.1 M
3-Hydroxybutyrate 25 mM
NAD⁺ 1.8mM

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

2.3 ml Tris-HCl buffer, pH 8.5 (A)
0.5 ml Substrate solution (B)
0.2 ml NAD⁺ solution (C)


2. Add 0.1ml 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°C and calculate the ΔOD per minutes 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 0.1-0.5U/ml with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula :

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

Volume activity (U/ml) =                                                               =ΔOD/min×4.98×df

6.22×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (3.1ml)
Vs
: Sample volume (0.1ml)
6.22
: Millimolar extinction coefficient of NADH at 340nm (㎠/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(%) Substrate Relative activity(%)
DL-3-Hydroxybutyrate 100 sec-Butyl alcohol 0
3-Hydroxypropionate 0.2 Gluconate 0
DL-Lactate 0
 Glycolate 0
Glycerate 0
 NAD⁺ 100
2-Hydroxybutyrate 0 NADP⁺ 1.6
L-Malate
 0  
D,L-Malate 0    
Table 2. Effect of Various Chemicals on D-3-Hydroxybutyrate dehydrogenase
[The enzyme dissolved in 50 mM K-phosphate buffer, pH 6.5(10U/ml) was incubated at 25°C for 1hr.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 NaF 2 92
MgCl₂ 2 109 NaN₃
 20 88
CaCl₂ 2 116 EDTA 5 38
FeCl₃
2 14 o-Phenanthroline 2 94
CoCl₂ 2 109 α,α′-Dipyridyl 1 89
MnCl₂ 2 118 IAA 2 58
ZnSO₄ 2 97 NEM 2 0
NiCl₂ 2 103 Hydroxylamine 2 100
CuSO₄ 2 94 Triton X-100 0.10% 106
AgNO₃ 2 94 Brij 35 0.10% 103
MIA 2 73 Tween 20 0.10% 103
      Span 20 0.10% 103
   
Na-cholate 0.10% 103
      SDS 0.05% 35
      DAC 0.05% 95

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



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)

 

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