臨床検査において、尿酸の測定に利用されます。
| 由来 | recombinant E. coli |
|---|---|
| 系統名 | Urate : oxygen oxidoreductase |
| EC 番号 | 1.7.3.3 |
| 反応式 | Urate + H2O + O2 →→→ Unidentified products + H2O2 ↓ CO2 + Allantoin (racemic mixture) |
Uricase (U-TE) ウリカーゼ(U-TE)
- 臨床診断用酵素
仕様
| Appearance | light brownish lyophilizate | |
|---|---|---|
| Activity | ≧4.0 U/mg | |
| Contaminants | Catalase ≦1.0 U/U% | |
| Stabilizer | sucrose, citrate, ethylenediaminetetraacetic acid (EDTA) | |
| Storage condition | below -20℃ |
特性
| Molecular weight | ca. 90 kDa (gel filtration) |
|---|---|
| Structure | 2 subunits of 35 kDa (SDS-PAGE) |
| Michaelis constant | 1.1×10-5M (uric acid) |
| pH Optimum | 8.5 (Fig. 1) |
| pH Stability | 7.0–11.0 (Fig. 2) |
| Optimum temperature | 45℃ (Fig. 3) |
| Thermal stability | below 55℃ (Fig. 4) |
| Stability (liquid form) | stable at 37℃ for at least ten days (Fig. 5) |
| Stability (powder form) | stable at 30℃ at least three weeks (Fig. 6) |
| Inhibitors | Hg2+，Ag+ |
アプリケーション
臨床検査において、尿酸の測定に利用されます。
分析手順
Principle
The disappearance of uric acid is measured spectrophotometrically at 290 nm.
Definition of unit
One unit (U) is defined as the amount of enzyme which oxidizes 1 µmol of uric acid per min at 25℃ and pH 8.5 under the conditions described below.
Reagents
- KOH solution, 20%: 20 g of KOH/100 ml of distilled water.
- Uric acid solution, 0.001%: dilute the stock solution (0.01%) to 10-fold volume with enzyme dilution buffer (Reagent C) (prepare freshly). Stock solution: 10 mg of uric acid/100 ml of enzyme dilution buffer (Reagent C).
- Enzyme dilution buffer: dissolve 3.09 g of boric acid, 0.37 g of EDTA-Na2-2H2O and 0.01 g of Triton X-100 in 800 ml of distilled water, adjust to pH 8.5 with 2 N NaOH and dilute with distilled water to 1000 ml.
Sample: dissolve the lyophilized enzyme to a volume activity of 0.01–0.02 U/ml with ice-cold enzyme dilution buffer (Reagent C) immediately before measurement.
Procedure
- Pipette the following reagent into a test tube.
2.0 ml Uric acid solution (Reagent B) 0.5 ml PDistilled water - Equilibrate at 25℃ for about 5 min.
- Add 0.5 ml of sample and incubate for 5 min at 25℃.
- Add 0.2 ml of KOH solution (Reagent A) to stop the reaction.
- Read the absorbance at 290 nm in a cuvette (light path: 1 cm) (AS).
The blank solution is prepared by reversing the sequence of addition of sample and KOH solution (Reagent A) (A0).
Calculation
Activity can be calculated by using the following formula:
実験データ
参考文献
Koyama, Y. et al., J. Biochem., 120, 969–973 (1996).