XANTHINE OXIDASE from Microorganism
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% | |
Purine-nucleoside phosphorylase | ≤5.0×10-3% | |
Stabilizers | Sodium glutamate, BSA |
Stability | Stable at −20℃ for at least one year (Fig.1) |
---|---|
Molecular weight | approx. 160,000 |
Isoelectric point | 4.0±0.1 |
Michaelis constant | 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℃ (Fig.4) |
pH Stability | pH 6.5−9.0 (25℃, 15hr) (Fig.5) |
Thermal stability | below 55℃ (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).
The appearance of uric acid is measured at 293nm by spectrophotometry.
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 |
1.Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.
2.24ml | Tris-HCl buffer, pH 7.5 | (A) |
---|---|---|
0.08ml | Xanthine solution | (C) |
0.08ml | Oxonic acid solution | (D) |
Concentration in assay mixture | |
---|---|
Tris-HCl buffer | ca.89.6 mM |
Xanthine | 0.32mM |
Oxonic acid | 32 μM |
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℃, 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 :
Volume activity (U/ml) =
ΔOD/min (ΔOD test−ΔOD blank)×Vt×df
12.5×1.0×Vs
= ΔOD/min×2.0×df
Weight activity (U/mg) = (U/ml)×1/C
Vt | : Total volume (2.5ml) |
Vs | : Sample volume (0.1ml) |
12.5 | : Millimolar extinction coefficient of uric acid under the assay condition (㎠/micromole) |
1.0 | : Light path length (cm) |
df | : Dilution factor |
C | : Enzyme concentration in dissolution (c mg/ml) |
Substrate(100mM) | Relative activity(%) |
---|---|
Xanthine | 100 |
Hypoxanthine | 18.7 |
Purine | 6.4 |
Guanine | 18.3 |
Adenosine | 0 |
[The enzyme dissolved in 50mM Tris-HCl buffer, pH 7.5 (2U/ml) was incubated with each chemical at 25℃ for 2hr.]
Chemical | Concn.(mM) | Residual activity(%) |
---|---|---|
None | - | 100 |
Metal salt | 2.0 | |
MgCl2 | 78 | |
CaCl2 | 96 | |
Ba(OAc)2 | 102 | |
FeCl3 | 85 | |
CoCl2 | 100 | |
MnCl2 | 102 | |
ZnSO4 | 62 | |
CdCl2 | 34 | |
NiCl2 | 91 | |
CuSO4 | 18 | |
PbCl2 | 9.7 | |
AgCl | 0 | |
HgCl2 | 1.2 |
Chemical | Concn.(mM) | Residual activity(%) |
---|---|---|
PCMB | 2.0 | 24 |
MIA | 2.0 | 1.1 |
NaF | 2.0 | 14 |
NaN3 | 20 | 25 |
EDTA | 5.0 | 95 |
o-Phenanthroline | 2.0 | 89 |
α,α′-Dipyridyl | 1.0 | 81 |
Borate | 20 | 89 |
IAA | 2.0 | 24 |
NEM | 2.0 | 91 |
Triton X-100 | 0.1% | 99 |
Brij 35 | 0.1% | 50 |
Tween | 0.1% | 106 |
Span 20 | 0.1% | 110 |
Na-cholate | 0.1% | 115 |
SDS | 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.
Fig.1. Stability (Powder form)
(kept under dry conditions)
Fig.2. Stability (Liquid form)
in 50mM buffer solution 4℃,pH6.8 enzyme concn.:60U/ml
Fig.3. pH-Activity
37℃ in 4min-reaction in 50mM buffer solution
●:pH6.0-8.2 K-phosphate
○:pH7.5-8.5 Tris-HCI
Fig.4. Thermal activity
(in 50mM Tris-HCl buffer, pH8.0)
Fig.5. pH-Stability
25℃, 15hr with 50mM buffer solution
●:pH4.0-6.0 acetate
○:pH6.0-8.0 K-phosphate
■:pH8.0-9.0 Tris-HCI
□:pH9.0-10.0 Na-K carbonate
Fig.6. Thermal stability
30min-treatment with 50mM Tris-HCI buffer,pH8.0
1. 原理
尿酸の生成量を293nmにおける吸光度の変化で測定する。
2.定義
下記条件で1分間に1マイクロモルの尿酸を生成する酵素量を1単位(U)とする。
3.試薬
酵素溶液:酵素標品を予め氷冷した50mM Tris-HCl緩 衝液,pH7.5で溶解し,同緩衝液で0.1- 0.2U/mlに希釈して氷冷保存する。
4.手順
1.下記反応混液をキュベット(d=1.0cm)に調製し,37℃ で約5分間予備加温する。
2.24ml | Tris-HCl緩衝液 | (A) |
0.08ml | キサンチン水溶液 | (C) |
0.08ml | オキソン酸・K水溶液 | (D) |
2.酵素溶液0.1mlを添加し,ゆるやかに混和後,水を対照に37℃に制御された分光光度計で293nmの吸光度変化を求める(ΔODtest)。
3.盲検は反応混液①に酵素溶液の代わりに酵素希釈液(50mM Tris-HCl緩衝液, pH7.5)を0.1ml加え,上記同様に操作を行って,1分間当たりの吸光度変化を求める(ΔODblank)。
5.計算式
U/ml =
ΔOD/min (OD test−OD blank)×2.5(ml)×希釈倍率
12.5×1.0×0.1(ml)
= ΔOD/min×2.0×希釈倍率 | |
U/mg | = U/ml×1 / C |
12.5 | : 尿酸のミリモル分子吸光係数 (cm2/micromole) |
1.0 | : 光路長(cm) |
C | : 溶解時の酵素濃度(c mg/ml) |
各種製品に関するご質問・ご相談はこちらよりお問い合わせください。