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ASO-312<br />
TOYOBO ENZYMES<br />
(Diagnostic Reagent Grade)<br />
<strong>ASCORBATE</strong> <strong>OXIDASE</strong><br />
from Cucurbita sp.<br />
L-Ascorbate : Oxygen oxidoreductase (EC 1. 10. 3. 3)<br />
L-Ascorbic acid 1 /2O 2<br />
Dehydroascorbic acidH 2 O<br />
PREPARATION and SPECIFICATION<br />
Appearance<br />
: Light blue amorphous powder, lyophilized<br />
Activity<br />
: Grade40U/mg-solid or more<br />
Contaminants : Catalase ≤1.010 1 %<br />
Phosphatase ≤2.010 2 %<br />
Stabilizers<br />
: BSA, sugars<br />
PROPERTIES<br />
Stability : Stable at 20 for at least one year Fig.1<br />
Michaelis constant<br />
: 3.010 4 M(Ascorbate)<br />
Inhibitors<br />
: cyanide, Na 2 S, diethyldithiocarbamate (Na)<br />
Optimum pH : 6.0 Fig.3<br />
Optimum temperature : 60 Fig.4<br />
pH Stability : pH 6.010.0 (25, 20hr) Fig.5<br />
Thermal stability : below 45 (pH 7.0, 30min) Fig.6<br />
Substrate specificity<br />
: The enzyme oxidizes ascorbic acid and several ascorbic acid<br />
derivatives.<br />
Effect of various chemicals : (Table 1)<br />
APPLICATIONS<br />
This enzyme is useful for enzymatic determination of ascorbic acid and for eliminating the interference<br />
of ascorbic acid in clinical analysis.<br />
33
ASO-312<br />
ASSAY<br />
Principle:<br />
Ascorbic acid 1 ascorbate oxidase<br />
/ 2 O 2<br />
Dehydroascorbic acidH 2 O<br />
The disappearance of ascorbic acid is measured at 245nm by spectrophotometry.<br />
Unit definition:<br />
One unit causes the decrease of one micromole of ascorbic acid per minute under the conditions described below.<br />
Method:<br />
Reagents<br />
A. Ascorbic acid solution 1.0mM Dilute the stock solution (10mM) to 10-fold volume with 0.2 M KH 2 PO 4<br />
solution containing 1.0mM EDTA.(Should be prepared fresh) Stock solution :<br />
176mg L-ascorbic acid (MW176.13)/100ml of 1.0mM HCl solution containing<br />
1.0mM EDTA (Stable for one month if stored at 05)<br />
B. Na 2 HPO 4 solution<br />
C. HCl solution<br />
D. Enzyme diluent<br />
10mM<br />
0.2N<br />
10mM Na 2 HPO 4 solution containing 0.05% BSA (Should be prepared fresh)<br />
Procedure<br />
1. Prepare the following reaction mixture in a test tube and<br />
Concentration in assay mixture<br />
equilibrate at 30 for about 5 minutes.<br />
KH 2 PO 4<br />
82 mM<br />
0.5ml Substrate solution (A) Na 2 HPO 4<br />
5.5 mM<br />
0.5ml Na 2 HPO 4 solution (B) Ascorbic acid<br />
0.45 mM<br />
(pH of the reaction mixture should be 5.6.)<br />
EDTA<br />
0.45 mM<br />
2. Add 0.1ml of the enzyme solution and mix.<br />
BSA<br />
45.4g/ml<br />
3. After exactly 5 minutes at 30, add 3.0ml of HCl solution (C) to stop the reaction and measure the optical<br />
density at 245nm against water (OD test).<br />
At the same time, prepare the blank by first mixing the reaction mixture with 3.0ml of HCl solution (C) after<br />
5 min-incubation at 30, followed by the addition of the enzyme solution (OD blank).<br />
<br />
Dissolve the enzyme preparation in ice-cold distilled water (more than 60U/ml) and dilute to 0.150.25U/ml<br />
with ice-cold enzyme diluent (D), immediately before assay.<br />
Calculation<br />
Activity can be calculated by using the following formula<br />
Volume activity (U/ml) <br />
OD (OD testOD blank)Vtdf<br />
10.01.0tVs<br />
OD0.820df<br />
Weight activity (U/mg)(U/ml)1/C<br />
Vt Total volume (4.1ml)<br />
Vs Sample volume (0.10ml)<br />
10.0 Millimolar extinction coefficient of ascorbic acid under the assay condition at pH 1.0<br />
(F/micromole)<br />
1.0 Light path length (cm)<br />
t Reaction time (5 minutes)<br />
df Dilution factor<br />
C Enzyme concentration in dissolution (c mg/ml)<br />
REFERENCES<br />
1) T.Nakamura, N.Makino and Y.Ogura; J.Biochem., 64, 189 (1968).<br />
2) V.Ts.Aikazyan and R.M.Nalbandyan; FEBS LETTERS, 104, 127 (1979).<br />
3) G.A.White and F.G.Smith; Nature, 190, 187 (1961).<br />
34
ASO-312<br />
Table 1. Effect of Various Chemicals on Ascorbate oxidase<br />
The enzyme dissolved in 10mM K-phosphate buffer, pH 7.0 contg. 0.2% BSA (55U/ml) was incubated with<br />
each chemical at 25]<br />
Chemical<br />
None<br />
Metal salt<br />
MgCl 2<br />
CaCl 2<br />
Ba(OAc) 2<br />
FeCl 3<br />
CoCl 2<br />
MnCl 2<br />
ZnCl 2<br />
CdCl 2<br />
NiCl 2<br />
CuSO 4<br />
Pb(OAc) 2<br />
AgNO 3<br />
HgCl 2<br />
2-Mercaptoethanol<br />
PCMB<br />
Residual<br />
Concn.(mM)<br />
activity<br />
100%<br />
2.0<br />
2.0<br />
2.0<br />
98<br />
98<br />
101<br />
104<br />
102<br />
101<br />
97<br />
102<br />
101<br />
20<br />
97<br />
1.2<br />
0<br />
99<br />
100<br />
Chemical<br />
MIA<br />
NEM<br />
IAA<br />
Hydroxylamine<br />
EDTA<br />
o-Phenanthroline<br />
,-Dipyridyl<br />
Borate<br />
NaF<br />
NaN 3<br />
Triton X-100<br />
Brij 35<br />
Tween 20<br />
Span 20<br />
Na-cholate<br />
SDS<br />
DAC<br />
Concn.(mM)<br />
2.0<br />
2.0<br />
2.0<br />
2.0<br />
5.0<br />
2.0<br />
1.0<br />
50<br />
2.0<br />
2.0<br />
0.10%<br />
0.10%<br />
0.10%<br />
0.10%<br />
0.10%<br />
0.05%<br />
0.05%<br />
Ac, CH 3 CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate;<br />
IAA, lodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate;<br />
DAC, Dimethl-benzyl-alkyl-ammonium-chloride.<br />
Residual<br />
activity<br />
99%<br />
99<br />
99<br />
104<br />
99<br />
77<br />
98<br />
103<br />
103<br />
103<br />
106<br />
107<br />
111<br />
104<br />
107<br />
106<br />
98<br />
100<br />
100<br />
100<br />
Residual Activity,%<br />
50<br />
-20<br />
Relative Activity<br />
50<br />
Residual Activity,%<br />
50<br />
0<br />
0<br />
5 10 15<br />
20<br />
0<br />
0<br />
3<br />
4 5 6 7 8 10<br />
0<br />
0<br />
3<br />
4 5 6 7 8 9 10 11<br />
Period (months)<br />
pH<br />
pH<br />
Fig.1. Stability (Powder form)<br />
kept under dry conditions<br />
Fig.3. pH-Activity<br />
30 in 0.1 M buffer solution, pH4.0-6.0,<br />
acetate; pH6.0-8.0<br />
phosphate;pH8.0-10.0,borate<br />
Fig.5. pH-Stability<br />
25,20hr-treatment with in 0.1 M<br />
buffer solution:pH4.0-6.0,<br />
acetate; pH6.0-8.0, phosphate;<br />
pH8.0-10.0,borate<br />
100<br />
100<br />
100<br />
Residual Activity,%<br />
50<br />
0<br />
0<br />
-311<br />
-312<br />
2 4 6<br />
8<br />
Relative Activity<br />
50<br />
0<br />
0<br />
20<br />
30 40 50 60 70<br />
Residual Activity,%<br />
50<br />
0<br />
0<br />
20<br />
40<br />
60<br />
80<br />
Period (days)<br />
Fig.2. Stability (Liquid form)<br />
40 in 0.1 M PIPES buffer solution, pH7.0<br />
(contg.0.05% BSA)<br />
Temperature, <br />
Fig.4. Temperature activity<br />
in 0.33M phosphate buffer pH5.6<br />
Temperature, <br />
Fig.6. Thermal stability<br />
30min-treatment with 10mM<br />
phosphate buffer,pH 7.0<br />
(contg.0.2% BSA)<br />
enzyme concn.: 15U/ml<br />
35
ASO-312<br />
<br />
<br />
2<br />
ascorbate oxidase<br />
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P <br />
P 2 4 <br />
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P<br />
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F<br />
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36