D-LACTATE DEHYDROGENASE from Microorganism

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅡ 400U/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 (by gel filtration)
Isoelectric point:  4.0
Michaelis constant:  1.6×10⁻⁴M (pyruvate, pH 7.0)
Inhibitors :  Ag⁺, Hg⁺⁺, SH-reagents
Optimum pH :  6.0-7.0(Fig.3)
Optimum temperature :  35-40°C(Fig.4)
pH Stability :  pH 5.0-9.0 (25°C, 48hr)(Fig.5)
Thermal stability :  below 45°C (pH 7.0, 15min)(Fig.6)
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.

LCD-211

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 H₂O](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 0.098mM
BSA 16.4µg/mM

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

3.0 ml Substrate solution (A)
2.0 ml K-Phosphate buffer, pH 7.4 (B)
3.0 ml NADH solution (C)
22..0 ml 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 ΔOD per minute 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 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×Vs


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 Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 PCMB 2.0 89.3
Metal salt 2.0   MIA
2.0 0.1
MgCl₂   93.0 NaF 2.0 98.3
CaCl₂
   99.8 NaN₃ 20 94.6
Ba(OAc)₂   98.1 EDTA 5.0 99.2
FeCl₂   87.9
o-Phenanthroline 2.0 95.3
CoCl₂   91.5 α,α′-Dipyridyl 1.0 93.7
MnCl₂   92.2 Borate 50 95.6
ZnSO₄   89.6 IAA 2.0 33.6
Cd(OAc)₂   91.3 NEM 2.0 97.7
NiCl₂   92.6 Hydroxylamine 2.0 95.6
CuSO₄   93.8 Triton X-100 0.10% 121
Pb(OAc)₂   93.2
Brij 35 0.10% 116
AgNO₃   73.0 Tween 20 0.10% 117
HgCl₂   0 Span 20 0.10% 105
      Na-cholate 0.10% 112
      SDS 0.05% 109
      DAC 0.05% 52.0

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

To get a quote, contact us at info@toyobousa.com, or INQUIRY.