XANTHINE OXIDASE from Microorganism


Xanthine:oxygen oxidoreductase (EC

Hypoxanthine + O₂ + H₂O                      Xanthine + H₂O₂
Xanthine + O₂ + H₂O                       Uric acid + H₂O₂

Appearance Reddish brown amorphous powder, lyophilized
Activity GradeⅡ 10U/mg-solid or more
Contaminants Catalase ≤5%
Adenosine deaminase ≤1.0×10⁻³%
Uricase ≤1.0×10⁻³%
Phosphatase ≤1.0×10⁻³%
Purine-nucleoside phosphorylase ≤5.0×10⁻³%
Stabilizers Sodium glutamate, BSA
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight approx. 160,000
Isoelectric point 4.0±0.1
Michaelis constants 4.5×10⁻⁵M (Xanthine)
7.6×10⁻⁵M (Hypoxanthine)
Inhibitors Reducing agents, Hg⁺⁺, Ag⁺,MIA
Optimum pH 7.5-8.0(Fig.3)
Optimum temperature 65°C(Fig.4)
pH Stability pH 6.5-9.0 (25°C, 15hr)(Fig.5)
Thermal stability below 55°C (pH 8.0, 30min)(Fig.6)
Substrate specificity (Table 1)
Effect of various chemicals (Table 2)


This enzyme is useful for enzymatic determination of inorganic phosphorus, 5'-nucleotidase and adenosine deaminase when coupled with Purine-nucleoside phosphorylase (PNP-311) and uricase (UAO-201, UAO-211).



xanthine oxidase

Xanthine+O₂+H₂O                                                   Uric acid+H₂O₂

The appearance of uric acid is measured at 293nm by spectrophotometry.

Unit definition:

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


A. Tris-HCl buffer, pH 7.5 0.1M
B. Sodium hydroxide solution 0.025M
C. Xanthine solution 10mM[Dissolve 15.2mg of xanthine (MW=152.11) in 10ml solution (B)]
D. Oxonic acid potassium salt solution 1mM (Dissolve 9.75mg of oxonic acid・K salt in 50ml H₂O)
E. Enzyme diluent 50mM Tris-HCl buffer, pH 7.5


Concentration in assay mixture
Tris-HCl buffer ca.89.6 mM
Xanthine 0.32mM
Oxonic acid 32 µM

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

2.24ml Tris-HCl buffer, pH 7.5 (A)
0.08ml Xanthine solution (C)
0.08ml Oxonic acid solution (D)

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

3. Record the increase in optical density at 293nm 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 (E) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E), dilute to 0.1−0.2U/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×2.0×df


Weight activity (U/mg)=(U/ml)×1/C

: Total volume (2.5ml)
: Sample volume (0.1ml)
: Millimolar extinction coefficient of uric acid under the assay condition (㎠/micromole)
: Light path length (cm)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)

Table 1. Substrate Specificity of Xanthine oxygen (color-system)
Substrate(100mM) Relative activity
Xanthine 100
Hypoxanthine 18.7
Purine 6.4
Guanine 18.3
Adenosine 0
Table 2. Effect of Various Chemicals on Xanthine oxygen
[The enzyme dissolved in 50mM Tris-HCl buffer, pH 7.5 (2U/ml) was incubated with each chemical at 25°C for 2hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100 PCMB 2.0 24
Metal salt
2.0   MIA 2.0 1.1
MgCl₂   78
NaF 2.0 14
  96 NaN 20 25
Ba(OAc)₂   102 EDTA 5.0 95
FeCl₃   85 o-Phenanthroline
2.0 89
CoCl₂   100 α,α′- Dipyridyl 1.0 81
MnCl₂   102 Borate 20 89
ZnSO₄   62 IAA 2.0 24
CdCl₂   34 NEM 2.0 91
NiCl₂   91 Triton X-100 0.1% 99
CuSO₄   18 Brij 35 0.1% 50
PbCl₂   9.7 Tween 20 0.1% 106
AgCl   0
Span 20 0.1% 110
HgCl₂   1.2 Na-cholate 0.1% 115
0.05% 111
DAC 0.05% 78

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

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