PYRUVATE OXIDASE from Microorganism


Appearance Yellowish amorphous powder, lyophilized
Activity GradeⅢ 1.5U/mg-solid or more
Contaminants ATPase ≤5.0×10⁻²%
GOT, GPT ≤5.0×10⁻²%
Stabilizers Sugars, FAD
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 260,000
Isoelectric point 4.3
Michaelis constant 3.4×10⁻⁴M (Pyruvate)
Inhibitors Fe⁺⁺,Zn⁺⁺,Cu⁺⁺,Ag⁺,Hg⁺⁺
Optimum pH 5.7(Fig.2)
Optimum temperature 65°C(Fig.3)
pH Stability pH 5.7-6.5 (25°C, 20hr)(Fig.4)
Thermal stability below 45°C (pH 6.0, 15min)(Fig.5)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)


This enzyme is useful for enzymatic determination of pyruvate, GOT, GPT in clinical analysis.



pyruvate oxidase

Pyruvate+Pi+O₂+H₂O                                                   Acetylphosphate+CO₂+H₂O₂

TPP, FAD, Mg++


2H₂O₂+4-Aminoantipyrine+EHSPT                                                   Quinoneimine dye+4H₂O

The appearance of quinoneimine dye is measured at 550nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of hydrogen peroxide (half a micromole of quinoneimine dye) per
minute under the conditions described below.


A. Pyruvate solution 0.3M[378mg of Pyruvate・K salt (MW=126.15)/10ml of H₂O]
B. K-phosphate buffer, pH 5.9 0.15M
C. 4-Aminoantipyrine solution 0.15% (150mg of 4-Aminoantipyrine/100ml of H₂O)
D. EHSPT (TOOS) solution
0.3%[300mg of EHSPT (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)
/100ml of H₂O]
E . TPP solution 3mM[13.8mg of TPP (Thiamine pyrophosphate)(MW=460.77)/10ml of H₂O]
F . FAD solution 0.15mM [1.3mg of FAD・2Na salt (MW=865.55)/10ml of H₂O]
G. EDTA solution 15mM [590mg of EDTA・2Na salt (MW=394.22)/100ml of H₂O]
H. MgSO₄ solution 0.15M [3.4g of MgSO₄・7H₂O(246.48)/100ml of H₂O]
I . Peroxidase solution 50U/ml [45mg of peroxidase (110purpurogallin units/mg)/100ml of H₂O]
J . Enzyme diluent   50mM K-phosphate buffer, pH 5.7


Concentration in assay mixture
Pyruvate 48 mM
K-phosphate buffer 50 mM
4-Aminoantipyrine 0.48mM
EHSPT 0.58mM
TPP 0.19mM
FAD 0.01mM
EDTA 0.97mM
MgSO₄ 9.7 mM
Peroxidase ca.4.8 U/ml

1. Prepare the following working solution in a brownish bottle and store on ice.

10ml K-phosphate buffer, pH 5.9 (B)
2ml 4-Aminoantipyrine solution (C)
2ml EHSPT solution (D)
2ml TPP solutionn (E)
2ml FAD solution (F)
2ml EDTA solution (G)
2ml MgSO₄ solution (H)
3ml Peroxidase (I)

2. Pipette 2.5ml of working solution into a cuvette (d=1.0cm), add 0.5ml of pyruvate solution (A), and equilibrate at 37°C for about 5minutes.

3. Add 0.1ml of the enzyme solution* and mix by gentle inversion.

4. Record the increase in optical density at 550nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37°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.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (J), dilute to 0.1−0.5U/ml with the same buffer and store on ice.


Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD/min×1.68×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (3.10ml)
: Sample volume (0.10ml)
: Millimolar extinction coefficient of quinoneimine dye under the assay condition (㎠/micromole)
: Factor based on the fact that one mole of H₂O₂ produces half a mole of quinoneimine dye.
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. L.P.Hager, D.M.Geller and F.Lipman; Fed.Proc.,13, 734 (1954).
  2. B.Sedewitz, K.H.Schleifer and F.Gotz; J.Bacteriol,160, 273 (1984).
  3. B.Sedewitz, K.H.Schleifer and F.Gotz; J.Bacteriol,160, 462 (1984).
Table 1. Substrate Specificity of Pyruvate oxidase
Substrate(50mM) Relative activity(%) Substrate(50mM) Relative activity(%)
Pyruvate 100 Acetate 0
α-Ketobutyrate 5.8 Acetoacetate 0
α-Ketoglutarate 0 L-Alanine 0
Oxaloacetate 0 L-Aspartate 0
DL-Lactate 0    

Table 2. Effect of Various Chemicals on Pyruvate oxidase
[The enzyme dissolved in 50mM K-phosphate, pH 6.0 (10U/ml) was incubated with each chemical at 25°C for 1hr]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 NaF 2.0 100
Metal salt 2.0   NaN₃ 20 94
  96 EDTA 5.0 107
CaCl₂   93 o-Phenanthroline 2.0 97
Ba(OAc)₂   97
α,α′-Dipyridyl 1.0 95
FeCl₃   8.4 Borate 50 102
CoCl₂   84
IAA 2.0 102
MnCl₂   76 NEM 2.0 104
ZnSO₄   48 Hydroxylamine 2.0 98
Cd(OAc)₂   86
Triton X-100 0.10% 143
NiCl₂   119
Brij 35 0.10% 133
CuSO₄   0.9
Tween 20 0.10% 146
Pb(OAc)₂   33
Span 20 0.10% 121
AgNO₃   0 Na-cholate 0.10% 116
HgCl₂   0 SDS 0.05% 85
PCMB 1.0 66 DAC 0.05% 53
MIA 2.0 96      

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

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