PREPARATION and SPECIFICATION
This enzyme is useful for enzymatic determination of creatinine when coupled with creatine amidohydrolase and sarcosine oxidase.
One unit (U) is defined as the amount of enzyme which produces 1 μmol of creatine per min under the conditions described below.
Reagent I: Potassium phosphate buffer, 0.3 M, pH 6.5.
Reagent II: Creatinine solution, 0.1 M.
Reagent III: Sodium carbonate solution, 4%.
Reagent IV: a-Naphthol solution, 2%.
Reagent V: Alkaline solution, 1.2% NaOH, 3.2% Na2CO3.
Reagent VI: Diacetyl solution, 0.05%.
Enzyme diluent: 5 mM Tris-HCl, pH 8.0 enzyme diluent.
- Pipette 0.1 ml of reagent I and 0.8 ml reagent II into a test tube and equilibrate at 37 ℃ for about 5 minutes.
- Add 0.1 ml the enzyme solution and mix.
- After 10 minutes at 37 ℃, add 2.0ml of reagent III to stop the reaction and cool in ice water.
- Pipette successively the following reagents into a new test tube
- Allow to stand for about 1 h at 25 °C and dilute by adding 2 ml of distilled water.
- Read the absorbance at 525 nm in a cuvette (light path: 1 cm) (AS).
The blank solution is prepared by reversing the sequence of addition of sample and sodium carbonate solution (Reagent III) (Ab).
1.0: Total volume (mL)
0.1: Enzyme volume (mL)
1.0: Light path length (cm)
10: Reaction time (10 minutes)
df: Dilution factor
C: Enzyme concentration (mg/mL)
0.0704: Millimolar extinction coefficient of creatine under the assay conditions (cm2/μmol)
- Suzuki, M. and Yoshida, M., Clin. Chim. Acta, 143, 147–155 (1984).