URICASE [UODNⅡ]
from Arthrobacter globiformis
(Urate: oxygen oxidoreductase, EC 1.7.3.3)
(Urate oxidase)
Uric Acid + O2 + 2 H2O → Allantoin + H2O2 + CO2
Preparation and Specification
- Appearance
- : White to off white amorphous powder, lyophilized
- Specific activity
- : More than 10 U/mg solid
Properties
- Molecular weight
- : 117 kDa (TSK gel G3000SWXL)
- Isoelectric point
- : pH 4.61
- Michaelis constants
- : Uric acid 1.3 × 10−4 M
- Optimum pH
- : 8.5–9.5Figure 1
- pH stability
- : 8.5–9.5 (37℃ , 60 min) Figure 2
- Thermal stability
- : Stable at 50℃ and below (pH 9.0, 10 min) Figure 3
- Storage stability
- : At least one year at −20℃Figure 4
Applications for Diagnostic Test
This enzyme is useful for enzymatic determination of uric acid.
| UODN Ⅱ | ||
| Uric Acid + O2 + 2 H2O | → | Allantoin + H2O2 + CO2 |
| POD | ||
| 2 H2O2 +4－AA+Phenol | → | Quinoneimine dye + 4H2O |
Fig.1 pH Optimum
〇: 3, 3-Dimethylglutarate
-NaOH buffer
△: Phosphate buffer
●: Ｔris-HCI buffer
▲: Borate buffer
-NaOH buffer
△: Phosphate buffer
●: Ｔris-HCI buffer
▲: Borate buffer
Fig.2 pH Stability
37℃, 60min
●: Phosphate buffer
■: Ｔris-HCI buffer
〇: Glycine-NaOH buffer
▲: Borate buffer
●: Phosphate buffer
■: Ｔris-HCI buffer
〇: Glycine-NaOH buffer
▲: Borate buffer
Fig.3 Thermal Stability
pH9.0 10 min.
20mM Borate buffer
20mM Borate buffer
Fig.4 Storage (lyophilized powder)
－20℃
Assay
Principle
The assay is based on the decrease in absorbance at 293 nm of uric acid which is oxidized in the following reaction:
| UODN Ⅱ | ||
| Uric acid+O2+2 H2O | → | Allantoin+H2O2+CO2 |
Unit definition
-
One unit is defined as the amount of enzyme which oxidizes 1 μmole of uric acid to allantoin per minute at 25℃ under the conditions specified in the assay procedure.
Reagents
- Reaction mixture (0.115 mM Uric acid)
Mix 1 ml of 3.57 mM uric acid and 30 ml of enzyme dilution buffer. - Enzyme dilution buffer
20 mM sodium tetraborate–HCl buffer pH 9.0 - Reagents
Sodium tetraboric acid (Na2B4O7・10H2O) :FUJIFILM Wako Pure Chemical CorporationUric acid: Tokyo Kasei Kogyo Co., Ltd. #U0018
Special grade #194–01415
Enzyme solution
-
Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.
Procedure
- Pipette accurately 3.10 ml of reaction mixture into a small test tube and preincubate at 25℃.
- After 3 min, add 20 μl of enzyme solution and mix to start the reaction at 25℃.
※ In the case of a test blank, add 20 μl of enzyme dilution buffer in place of enzyme solution. - After starting the reaction, measure the rate of decrease
- per minute in absorbance at 293 nm. The rate must be measured within the linear portion of the absorbance curve.
△ A/min = (Ab/min−As/min) ≦ 0.060 Abs/minAbsorbance sample : As/min blank : Ab/min
Calculation
Activity (U/mg of powder) = {(△A/min) /(12.6)}× 3.12/0.02 × 1/x| 12.6 : | millimolar extinction coefficient of uric acid at 293 nm
(cm2 / μmole) |
| 3.12 : | final volume (ml) |
| 0.02 : | volume of enzyme solution (ml) |
| X : | concentration of the sample in enzyme solution (mg/ml) |
Storage
-
Storage at − 20℃ in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition (Figure 4) .
References
- Bongaerts, G. P. A., Uitzerter, J., Brouns, R. and Vogeis, G.
D. (1978) Biochim. Biophys. Acta, 527, 348–358. - Bongaerts, G. P. A. and Vogeis, G. D. (1976) J. Bacteriol., 125, 689–697.
- Itaya, K., Yamamoto, T. and Fukumoto, J. (1967) Agric.
Biol. Chem., 31, 1256. - Nakagiri, Y. and Yamamoto, T. (1971) Eisei Kensa, 20, 751–759.
- Kageyama, N. et al. (1969) Eisei Kensa, 19, 338–342.
- Kageyama, N. et al. (1969) Eisei Kensa, 18, 59–63.
- Kageyama, N. (1972) Rinsho Kensa, 16, 891.
- Kageyama, N. (1971) Clin. Chem. Acta., 31, 421–426
- Kawashima, T. et al. (1980) Nihon Kagakukaishi, 10, 1542.
UODN Ⅱ活性測定法 (Japanese)
試薬液
- 反応試薬混合液 (0.115mM 尿酸)
3.57mM 尿酸溶液1ml と酵素溶解希釈用液30ml を混合する。 - 酵素溶解希釈用液
20mM 四ホウ酸ナトリウム−HCl 緩衝液 pH9.0 - 試薬
四ホウ酸ナトリウム (Na2B4O7・10H2O) ：富士フイルム和光純薬製 特級 #194–01415尿酸 (Uric acid) ：東京化成製 #U0018
酵素試料液
- 検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。
その液を酵素溶解希釈用液で適宜希釈する。
測定操作法
- 小試験管に反応試薬混合液3.10ml を正確に分注して25℃で予備加温する。
- 3 分経過後、酵素試料液20 μl を加えて混和し、25℃で反応を開始する。
※ 盲検は酵素試料液の代わりに酵素溶解希釈用液20μl を加える。 - 反応開始後、293nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
求められた吸光度を試料液はAs/min、盲検液は
Ab/min とする。
Δ A/min = (As/min−Ab/min) ≦ 0.060 Abs/min
計算
活性 (U/mg) = {(△ A/min)/ (12.6)}× 3.12/0.02 × 1/x| 12.6 : | 尿酸の293nm におけるミリモル分子吸光係数 (cm2 / μmole) |
| 3.12 : | 反応総液量 (ml) |
| 0.02 : | 反応に供した酵素試料液量 (ml) |
| X : | 酵素試料液中の検品濃度 (mg/ml) |
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