D-3-HYDROXYBUTYRATE DEHYDROGENASE from Pseudomonas sp.

HBD-301

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅢ 100U/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°C for at least Two years (Fig.1)
Molecular weight :  approx. 130,000 (by gel filtration)
Isoelectric point :  5.6±0.1
Michaelis constants :  4.2×10⁻⁴M (25°C, pH8.3), 7.0×10⁻⁴M(37°C, pH8.3)(D-3-Hydroxybutyrate)
4.9×10⁻⁵M (25°C, pH8.3), 7.2×10⁻⁵M (37°C, pH8.3)(NAD⁺)
8.1×10⁻⁵M (25°C, pH7.1), 2.4×10⁻⁴M (37°C, pH7.1)(Acetoacetate)
8.4×10⁻⁶M (25°C, pH7.1), 1.5×10⁻⁵M (37°C, pH7.1)(NADH)
Inhibitors :  PCMB, MIA, IAA, Ag⁺, Hg⁺⁺, SDS, DAC
Optimum pH :  8.3(Fig.3)
Optimum temperature :  55°C(Fig.4)
pH Stability :  pH 5.0-8.5 (25°C, 20hr)(Fig.5)
Thermal stability :  below 40°C (pH 6.5, 15min)(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:

D-3-hydroxybutyrate dehydrogenase

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


The appearance 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 340nm against water for 2 to 3 minutes in 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) by 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(%)
3-Hydroxybutyrate 100 sec-Butyl alcohol 0
3-Hydroxypropionate 0.14 Gluconate 0
Lactate 0
 Glycolate 0.04
Glycerate 0
    
2-Hydroxybutyrate 0 NAD⁺ 100
L-Malate
 0 NADP⁺ 4.74
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 2.0 100
Metal salt 2.0   NaN₃
 20 104
MgCl₂   105 EDTA 5.0 97
CaCl₂
   101 o-Phenanthroline 2.0 96
Ba(OAc)₂   98 α,α′-Dipyridyl 1.0 97
FeCl₃   101 Borate 50 103
CoCl₂   102 IAA 2.0 4
MnCl₂   103 NEM 2.0 59
ZnSO₄   100 Hydroxylamine 2.0 101
Cd(OAc)₂   100 Triton X-100 0.10% 113
NiCl₂   103 Brij 35
 0.10% 37
CuSO₄   83 Tween 20 0.10% 68
Pb(OAc)₂   96
 Span 20 0.10% 104
AgNO₃   2.5 Na-cholate 0.10% 107
HgCl₂   0 SDS
 0.05% 5
PCMB 2.0 0 DAC 0.05% 4
MIA 2.0 1      

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

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