D-LACTATE DEHYDROGENASE from Microorganism

LCD-221

(R)-Lactate : NAD⁺ oxidoreductase (EC 1. 1. 1. 28)
D-Lactate + NAD⁺ ⇆ Pyruvate + NADH + H⁺

PREPARATION and SPECIFICATION
Appearance:  White amorphos powder, lyophilized
Activity:  Grade II 900 U/mg-solid or more
Contaminants:  NADH oxidase ≤1.0×10⁻³%
Malate dehydrogenase ≤1.0×10⁻²%
GOT ≤5.0×10⁻³%
GPT ≤5.0×10⁻³%
Myokinase ≤1.0×10⁻²%
Pyruvate kinase ≤1.0×10⁻³%
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1)
Molecular weight :  approx. 140,000
Isoelectric point:  4.0
Michaelis constant:  6.4×10⁻⁴M (pyruvate, pH 7.0)
Inhibitors :  Ag²+, Hg²+, SH-reagents
Optimum pH :  5.0-7.0 (Fig.2)
Optimum temperature :  30-37°C(Fig.3)
pH Stability :  pH 5.0-9.0 (25°C, 48hr) (Fig.4)
Thermal stability :  below 45°C (pH 7.0, 15min) (Fig.5)
Effect of various chemicals :  (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of numerous metabolites, e.g. ATP, ADP, glucose, creatinine, pyruvate, lactate and glycerol, and of enzyme activities, e.g. GPT, PK, and CPK when coupled with the related enzymes.

ASSAY

Principle:

D-Lactate dehydrogenase

Pyruvate + NADH +H⁺                                                 ►D-Lactate+ NAD⁺

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

Unit definition:

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

Method:

Reagents
A. Pyruvate solution :  5.0mM [5.50mg sodium pyruvate (MW=110)/10ml of H₂O]
(Should be prepared fresh)
B. K-Phosphate buffer, pH 7.4 :  1.0M
C. NADH solution :  1.0mM [7.63mg NADH・ 2Na (MW=763)/10ml of H₂ O]
(Should be prepared fresh)
D. Enzyme diluent :  0.1M K-phosphate buffer, pH 7.4 contg. 0.1% of BSA

Procedure

Concentration in assay mixture
K-phosphate buffer 67 mM
Pyruvate 0.49 mM
NADH 0.098 mM
BSA 16.4µg/ml

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

3.0ml Substrate solution (A)
2.0ml K-Phosphate buffer, pH 7.4 (B)
3.0ml NADH solution (C)
22.0ml H2O (D)

2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 25° C 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 2 to 3 minutes in a spectrophotometer thermostated at 25°C, and calculate the ΔΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔΔOD blank) by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

* Dilute the enzyme preparation to 0.2-1.0U/ml with ice-cold enzyme diluent (D), immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min(ΔOD test–ΔOD blank)× Vt× df

Volume activity (U/ml) =                                                                      = ΔOD/min× 9.81× df

6.22× 1.0× Vss


Weight activity (U/mg)=(U/ml)×1/C

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

REFERENCES

  1. C.A.Loshon, R.B.McComb, L.W.Bond, G.N.Bowers, Jr.W.H.Coleman and R.H.Gwynn; Clin.Chem., 23, 1576 (1977).
  2. H.Taguchi, M.Machida, H.Matsuzawa and T.Ohta; Agric.Biol.Chem., 49(2), 359 (1985).
  3. F.Gasser, M.Doudoroff, and R.Contopoulos; J.Gen. Microbiol. 62, 241 (1970).
Table 1. Effect of Various Chemicals on D-Lactate dehydrogenase
[The enzyme dissolved in 0.1M K-phosphate buffer, pH 7.4 (20U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical Conc.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 MIA 2.0 92.4
Metal salt 2.0   NaF 2.0 98
MgCl₂   100 NaN₃ 20 97.2
CaCl₂
   96.3 EDTA 5.0 96.0
Ba(OAc)₂   95.8
 o-Phenanthroline 2.0 98.0
FeCl₃   94.4
 α,α′-Dipyridyl 1.0 97.1
CoCl₂   97.3 Borate 50 97.5
MnCl₂   96.9 IAA 2.0 91.6
Cd(OAc)₂   97.2 NEM 2.0 95.2
NiCl₂   95.5 Hydroxylamine 2.0 96.3
CuSO₄   94.3 Triton X-100 0.10% 105.5
Pb(OAc)₂   96.0 Brij 35 0.10% 104.1
AgNO₃   71.5 Tween 20 0.10% 106.3
      Span 20 0.10% 98.2
   
Na-cholate 0.10% 102.1
      SDS 0.05% 104.4
      DAC 0.05% 47.6

Ac, CH₃CO; MIA; monoiodoacetate; EDTA, ethylenediaminetetraacetate; IAA, iodoacetamide; NEM,
N-ethylmaleimide; SDS, sodium dodecyl sulfate; DAC, dimethylbenzylalkylammonium chloride.

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