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 × 10−4M
- 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

-NaOH buffer
△: Phosphate buffer
●: Tris-HCI buffer
▲: Borate buffer
Fig.2 pH Stability

●: Phosphate buffer
■: Tris-HCI buffer
〇: Glycine-NaOH buffer
▲: Borate buffer
Fig.3 Thermal Stability

20mM Borate buffer
Fig.4 Storage (lyophilized powder)

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/x12.6 : | 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/x12.6 : | 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) |