PREPARATION and SPECIFICATION
PROPERTIES
APPLICATIONS
This enzyme is useful for enzymatic determination of creatinine when coupled with creatine amidohydrolase and s-oxidase.
UNIT DEFINITION
One unit (U) is defined as the amount of enzyme which produces 1 μmol of creatine per min under the conditions described below.
REAGENTS PREPARATION
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.
PROCEDURE
- 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).
CALCULATION
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)
REFERENCES
1.D.Tsuru;NucleicAcidandAminoAcids,35,31(1977).
2.T.Yoshimoto,I.OkaandD.Tsuru;Arch.Biochem.Biophys.,177,508(1976).
3.T.Yoshimoto,I.OkaandD.Tsuru;J.Biochem.,79,1381(1976).
4.D.Tsuru;RinshoKensa,22,1331(1978).
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