The enzyme is useful for the determination of creatinine and creatine in clinical analysis.
| 由来 | recombinant E. coli |
|---|---|
| 系统名称 | Creatine amidinohydrolase |
| EC编号 | 3.5.3.3 |
| 反应式 | Creatine + H2O →→→ Sarcosine + Urea |
Creatinase (C2-AT) 60118 肌酸酶
- 临床检测用酶
规格
| Appearance | white to light yellow lyophilizate | |
|---|---|---|
| Activity | ≧9 U/mg | |
| Stabilizer | sucrose | |
| Storage condition | below -20℃ |
特性
| Molecular weight | ca. 80 kDa (gel filtration) |
|---|---|
| Structure | 2 subunits of 48 kDa (SDS-PAGE) |
| Michaelis constant | 1.2×10-2M (creatine) |
| pH Optimum | 7.0–9.0 (Fig. 1) |
| pH Stability | 4.0–11.0 (Fig. 2) |
| Optimum temperature | 45℃ (Fig. 3) |
| Thermal stability | below 53℃ (Fig. 4) |
| Stability (liquid form) | stable at 37℃ for at least two weeks (Fig. 5) |
| Stability (powder form) | stable at 30℃ for at least one month (Fig. 6) |
| Inhibitors | Hg2+ |
适用类型
The enzyme is useful for the determination of creatinine and creatine in clinical analysis.
ASSAY PROCEDURE
Principle
The appearance of urea is measured spectrophotometrically at 435 nm.
Definition of unit
One unit (U) is defined as the amount of enzyme which produces 1 μmol of urea per min at 37℃ and pH 7.7 under the conditions described below.
Reagents
- Potassium phosphate buffer, 0.3 M; pH 7.7: mix 0.3 M KH2PO4 solution and 0.3 M K2HPO4 solution to make a pH 7.7 solution.
- Creatine solution, 0.1 M: 1.49 g of creatine monohydrate/100 ml of distilled water.
- p-Dimethylaminobenzaldehyde (DMAB) solution, 0.87%: dissolve 2.0 g of DMAB in 100 ml of ethanol (99.5%) and add 15 ml of conc. HCl and 115 ml of distilled water.
- Enzyme dilution buffer: mix 10 mm KH2PO4 solution and 10 mM K2HPO4 solution to make a pH 8.0 solution and add 2-mercaptoethanol (0.16 ml/liter of the buffer).
Sample: dissolve the lyophilized enzyme to a volume activity of 1.4–2.8 U/ml with ice-cold enzyme dilution buffer (Reagent D) immediately before measurement.
Procedure
- Pipette the following reagents into a test tube.
0.1 ml Potassium phosphate buffer (Reagent A) 0.8 ml Creatine solution (Reagent B) - Equilibrate at 37℃ for about 5 min.
- Add 0.1 ml of sample and incubate for 10 min at 37℃.
- Add 2.0 ml of DMAB solution (Reagent C) and allow to stand for about 30 min at 25℃.
- Read the absorbance at 435 nm in a cuvette (light path: 1 cm) (AS).
The blank solution is prepared by reversing the sequence of addition of sample and DMAB solution (Reagent C) (A0).
Calculation
Activity can be calculated by using the following formula:
EXPERIMENTAL DATA
Line-up
- Creatininase (C1-E)
- Creatinase (C2-AE)
- Creatinase (C2-AT)
- Creatinase (C2-EP)
- Sarcosine oxidase (SOD-TE)
- Sarcosine oxidase (SOD-EP)
FAQ
- What precautions should be taken when handling the product after opening?
- How should C2-AT be stored?
- What stabilizers are used in C2-AT?
- How thermostable is C2-AT?
- What are the optimal pH and temperature for C2-AT?
- What is the molecular weight of C2-AT?
- What are the main applications of C2-AT?
参考文献
Suzuki, M., Medical Technology, 7, 945–950 (1979).
Suzuki, M. and Yoshida, M. (1984)
A new enzymatic determination of serum creatine
Clinica Chimica Acta, 140, 289–294.
Suzuki, M. and Yoshida, M. (1984)
A new enzymatic serum creatinine measurement based on an endogenous creatine-eliminating system
Clinica Chimica Acta, 143, 147–155.