ASCORBATE OXIDASE from Cucurbita sp.* ASO312

L-Ascorbate: Oxygen oxidoreductase (EC 1.10.3.3)
L-Ascorbic acid + ½O₂               ► Dehydroascorbic acid +H₂O

PREPARATION and SPECIFICATION
Appearance:  Light blue amorphous powder, lyophilized
Activity:  GradeIII 40U/mg-solid or more
Contaminants:  Catalase ≤1.0×10⁻¹ %
Phosphatase ≤2.0×10⁻² %
Stabilizers:  BSA, sugars
PROPERTIES
Stability:  Stable at -20°C for at least one year(Fig.1)
Michaelis constant:  3.0×10⁻⁴M(Ascorbate)
Inhibitors:  cyanide, Na₂S, diethyldithiocarbamate (Na)
Optimum pH:  6.0(Fig.3)
Optimum temperature: 60℃(Fig.4)
pH Stability:  pH 6.0-10.0 (25℃, 20hr)(Fig.5)
Thermal stability:   below 45℃ (pH 7.0, 30min)(Fig.6)
Substrate specificity:  The enzyme oxidizes ascorbic acid and several ascorbic acid
derivatives.
Effect of various chemicals:  (Table 1)

APPLICATIONS

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

ASO-312

ASSAY

Principle:

ascorbate oxidase

Ascorbic acid+½O₂                                 ►Dehydroascorbic acid+H₂O

 

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.

Method:

Reagents
A.Ascorbic acid solution:  1.0mM [Dilute the stock solution (10mM) to 10-fold volume with 0.2 M KH₂PO₄solution containing 1.0mM EDTA.](Should be prepared fresh) 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 prepared fresh)

Procedure

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℃, add 3.0ml of HCl solution (C) to stop the reaction and measure the optical density at 245nm against water (ΔODtest).
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℃, followed by addition of the enzyme solution (ΔOD 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.

Calculation

Activity can be calculated by using the following formula :

ΔOD (ΔOD blank−ΔOD 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

Vt
: Total volume (4.1ml)
Vs
: Sample volume (0.10ml)
10.0
: Millimolar extinction coefficient of ascorbic acid under the assay condition at pH 1.0 (㎠/micromole)
1.0
: Light path length (cm)
t
: Reaction time (5 minutes)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  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
activity		 Chemical Concn.(mM) Residual
activity
None − 100% MIA 2.0 99%
Metal salt 2.0   NEM 2.0 99
MgCl₂   98 IAA 2.0 99
CaCl₂ 98 Hydroxylamine 2.0 104
Ba(OAc)₂   101 EDTA 5.0 99
FeCl₃   104 o-Phenanthroline 2.0 77
CoCl₂   102 α,α'-Dipyridyl 1.0 98
MnCl₂   101 Borate 50 103
ZnCl₂   97 NaF 2.0 103
CdCl₂   102 NaN₃ 2.0 103
NiCl₂   101 Triton X-100 0.10% 106
CuSO₄   20 Brij 35 0.10% 107
Pb(OAc)₂   97 Tween 20 0.10% 111
AgNO₃   1.2 Span 20 0.10% 104
HgCl₂   0 Na-cholate 0.10% 107
2-Mercaptoethanol 2.0 99 SDS 0.05% 106
PCMB 2.0 100 DAC 0.05% 98

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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