ASCORBATE OXIDASE from Cucurbita sp.


L-Ascorbate: Oxygen oxidoreductase (EC
L-Ascorbic acid + ½O₂               Dehydroascorbic acid +H₂O

Appearance Light blue amorphous powder, lyophilized
Activity GradeIII 200U/mg -solid or more
Contaminants Catalase ≤1.0×10⁻¹ %
Phosphatase ≤2.0×10⁻² %
Stabilizers BSA, borate
Stability Stable at -20°C for at least One year (Fig.1)
Molecular weight 140,000
Isoelectric point 5.5± 0.2
Michaelis constant 2.7×10⁻⁴M (Ascorbate)
Inhibitors cyanide, Na₂S, diethyldithiocarbamate (Na)
Optimum pH pH 5.5-6.5(Fig.2)
Optimum temperature approx. 45℃(Fig.3)
pH Stability 6.0-10.0 (25℃, 20hr)(Fig.4)
Thermal stability below 45℃ (pH 7.0, 30min)(Fig.5)
Substrate specificity The enzyme oxidizes ascorbic acid and several ascorbic acid
Effect of various chemicals (Table 1)


This enzyme is useful for enzymatic determination of ascorbic acid and for eliminating the interference of ascorbic acid in clinical analysis.



ascorbate oxidase

Ascorbic acid+½O₂                                 Dehydroascorbic acid+HO


The disappearance of ascorbic acid is measured at 245nm by spectrophotometry.

Unit definition:

One unit causes the decrease of one micromole of ascorbic acid per minute under the conditions described below.


A.Ascorbic acid solution 1.0mM [Dilute the stock solution (10mM) to 10-fold volume with 0.2M KH₂PO₄solution containing 1.0mM EDTA.](Prepare freshly)
Stock solution: 176mg L-ascorbic acid (MW=176.13)/100ml of 1.0mM HCl solution containing 1.0mM EDTA (Stable for one month if stored at 0-5℃)
B. Na₂HPO₄ solution 10mM
C. HCl solution 0.2N
D. Enzyme diluent 10mM Na₂HPO₄ solution containing 0.05% BSA (Should be freshly prepared)


Concentration in assay mixture
KH₂PO₄ 82 mM
Na₂HPO₄ 5.5 mM
Ascorbic acid 0.45 mM
EDTA 0.45 mM
BSA 45.4µ g/ml

1. Prepare the following reaction mixture in a test tube and equilibrate at 30℃ for about 5 minutes.

0.5ml Substrate solution (A)
0.5ml Na₂HPO₄ solution (B)
(pH of the reaction mixture should be 5.6.)

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

3. After exactly 5 minutes at 30°C, add 3.0ml of HCl solution (C) to stop the reaction and measure the optical density at 245nm against water (OD test).

At the same time, prepare the blank by first mixing the reaction mixture with 3.0ml of HCl solution (C) after 5min-incubation at 30°C, followed by addition of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold distilled water (more than 60U/ml) and dilute to 0.15-0.25U/ml
with ice-cold enzyme diluent (D), immediately before assay.


Activity can be calculated by using the following formula :

ΔOD (OD blank−OD test) × Vt × df

Volume activity (U/ml) =                                                               =ΔOD × 0.820 × df

10.0 × 1.0 × t × Vs


Weight activity (U/mg) = (U/ml) × 1/C

: Total volume (4.1ml)
: Sample volume (0.1ml)
: Millimolar extinction coefficient of ascorbic acid under the assay condition at pH 1.0 (㎠/micromole)
: Light path length (cm)
: Reaction time (5 minutes)
: Dilution factor
: Enzyme concentration in dissolution (c mg/ml)


  1. T.Nakamura, N.Makino and Y.Ogura; J.Biochem., 64, 189 (1968).
  2. V.Ts.Aikazyan and R.M.Nalbandyan; FEBS LETTERS, 104, 127 (1979).
  3. G.A.White and F.G.Smith; Nature, 190, 187 (1961).

Table 1. Effect of Various Chemicals on Ascorbate oxidase
[The enzyme dissolved in 10mM K-phosphate buffer, pH 7.0 contg. 0.2% BSA (55 U/ml) was incubated with each chemical at 25℃ for 1hr.]
Chemical Concn.(mM) Residual
Chemical Concn.(mM) Residual
None 100% MIA 2.0 100%
Metal salt 2.0   NEM 2.0 98
MgCl₂   96 IAA 2.0 97
CaCl₂ 98 Hydroxylamine 2.0 99
Ba(OAc)₂   98 EDTA 5.0 100
FeCl₃   100 o-Phenanthroline 2.0 84
CoCl₂   55 α,α'-Dipyridyl 1.0 98
MnCl₂   100 Borate 50 99
ZnCl₂   100 NaF 2.0 98
CdCl₂   86 NaN₃ 2.0 95
NiCl₂   99 Triton X-100 0.10% 100
CuSO₄   80 Brij 35 0.10% 100
Pb(OAc)₂   100 Tween 20 0.10% 100
AgNO₃   15 Span 20 0.10% 100
HgCl₂   5.2 Na-cholate 0.10% 97
2-Mercaptoethanol 2.0 94 SDS 0.05% 100
PCMB 1.0 86 DAC 0.05% 99

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

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