臨床検査において、各種コレステロールの測定に利用されます。
| 由来 | recombinant E. coli |
|---|---|
| 系統名 | Cholesterol : oxygen oxidoreductase |
| EC 番号 | 1.1.3.6 |
| 反応式 | Cholesterol + O2 →→→ Cholest-4-en-3-one + H2O2 |
Cholesterol Oxidase (CHO-CE) 60265 コレステロールオキシダーゼ(CHO-CE)
- 臨床診断用酵素
仕様
| Appearance | yellow lyophilizate | |
|---|---|---|
| Activity | ≧5 U/mg | |
| Stabilizer | trehalose,sodium malate, sodium glutamate, ethylenediaminetetraacetic acid (EDTA) | |
| Storage condition | below -20℃ protected from light |
特性
| Molecular weight | ca. 58 kDa (gel filtration) |
|---|---|
| Structure | monomer of 55 kDa (SDS-PAGE) |
| Michaelis constant | 3.5×10-4 M (cholesterol) |
| pH Optimum | ca. 7.0 |
| pH Stability | 5.0–10.0 |
| Optimum temperature | 50℃ |
| Thermal stability | below 55℃ |
| Stability (liquid form) | stable at 37℃ for at least one week |
| Stability (powder form) | stable at 30℃ for at least one month |
| Inhibitors | ionic detergents, Ag+, Hg2+ |
アプリケーション
The enzyme is useful for the determination of cholesterol in clinical analysis.
分析手順
Principle
The appearance of quinoneimine dye is measured spectrophotometrically at 500 nm.
Definition of unit
One unit (U) is defined as the amount of enzyme which produces 1 μmol of hydrogen peroxide per min at 37℃ and pH 7.0 under the conditions described below.
Reagents
- Potassium phosphate buffer, 0.1 M; pH 7.0: mix 0.1 M KH2PO4 solution and 0.1 M K2HPO4 solution to make a pH 7.0 solution.
- Cholesterol solution, 0.5%: dissolve 500 mg of cholesterol in 5.0 ml of Triton X-100 with heating. Add 90 ml of distilled water to the hot cholesterol–Triton X-100 solution by slowly pouring. Stir and boil for 30–60 s. Cool under running water with gentle agitation. Dissolve 4.0 g of sodium cholate in the cholesterol–Triton X-100 solution and dilute with distilled water to 100 ml. This solution is stable for about one week at room temperature. If it becomes cloudy, warm sligtly with stirring until it becomes clear.
- 4-Aminoantipyrine (4-AA) solution, 1.76%: 1.76 g of 4-AA/100 ml of distilled water.
- Phenol solution, 6.0%: 6.0 g of phenol/100 ml of distilled water.
- Peroxidase (POD) solution, 150 U/ml: 75 mg of POD (200 guaiacol U/mg)/100 ml of potassium phosphate buffer (Reagent A).
- Enzyme dilution buffer: 20 mm potassium phosphate buffer, pH 7.0, containing 0.2% bovine serum albumin (BSA).
Sample: dissolve the lyophilized enzyme to a volume activity of 0.1–0.5 U/ml with ice-cold enzyme dilution buffer (Reagent F).
Procedure
- Pipette the following reagents into a cuvette (light path: 1 cm).
2.55 ml Potassium phosphate buffer (Reagent A) 0.20 ml Cholesterol solution (Reagent B) 0.05 ml 4-AA solution (Reagent C) 0.10 ml Phenol solution (Reagent D) 0.10 ml POD solution (Reagent E) - Equilibrate at 37℃ for about 5 min.
- Add 0.05 ml of sample and mix.
- Record the increase of absorbance at 500 nm in a spectrophotometer thermostated at 37℃, and calculate the ΔA per min using the linear portion of the curve (ΔAS).
The blank solution is prepared by adding enzyme dilution buffer (Reagent F) instead of sample (ΔA0).
Calculation
Activity can be calculated by using the following formula:
13.8 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm2/μmol)
1/2 : Factor based on the fact that 1 mol of hydrogen peroxide produces 1/2 mol of quinoneimine dye
df : Dilution factor
C : Content of cholesterol oxidase preparation in sample (mg/ml)
実験データ
FAQ
- CHO-CEの主な用途は何ですか？
- CHO-CEの分子量を教えてください。
- CHO-CEの至適pHと至適温度について教えてください。
- CHO-CEの熱安定性について教えてください。
- CHO-CEの安定化剤は何を使用していますか？
- CHO-CEの保存方法を教えてください。
- 開封後の取扱い方法に注意事項はありますか？
参考文献
- Shirokane, Y. et al., J. Ferment. Technol., 55, 337–346 (1977).
- Richmond, W., Clin. Chem., 19, 1350–1356 (1973).
- Flegg, H. M., Ann. Clin. Biochem., 10, 79–84 (1973).
- Allain, C. C. et al., Clin. Chem., 20, 470–475 (1974).
- Tarbutton, P. N. and Gunter, C. R., Clin. Chem., 20, 724–725 (1974).