CAH (Creatinine amidohydrolase)
This enzyme is useful for enzymatic determination of creatinine when coupled with creatine amidohydrolase and sarcosine oxidase.
Specification
|
Appearance |
White to slightly yellowish amorphous powder, lyophilized |
|
Purity(SDS-PAGE) |
≥90.0% |
|
Enzyme powder specific activity |
≥400.0 U/mg |
|
Catalase |
≤0. 1% |
|
ATPase |
≤0.001% |
|
Proteases |
≤0.001% |
|
Creatine amidinohydrolase |
≤0.001% |
Properties
|
EC number |
3.5.2.10 (Recombinant from microorganism) |
|
|
Molecular weight |
29 kDa (SDS-PAGE) |
|
|
Isoelectric point |
5.3 |
|
|
Michaelis constants |
5.0× 10-2 M (Creatinine), 8.0× 10-2 M (Creatine) |
|
|
Inhibitors |
Hg2+, Cu2+, Fe3+ |
|
|
Optimum pH |
7.0-8.0 |
Fig. 1 |
|
Optimum temperature |
65 ℃ |
Fig. 2 |
|
pH stability |
pH 5.5- 10.0 (25 ℃, 16 h) |
Fig. 3 |
|
Thermal stability |
Below 65 ℃ (pH 8.0, 30 min) |
Fig. 4 |
|
Storage stability |
At least one year at -25 ~ - 15 ℃ |
Fig. 5 |
|
Stabilizers |
BSA, sugar |
|
Figures
Assay Principle
The appearance of creatine is measured at 525 nm by spectrophotometry.
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.
Sample: dilute the enzyme to 2.0−4.0 U/ml with enzyme diluent.
Procedure
1.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.
2.Add 0. 1 ml the enzyme solution and mix.
3.After 10 minutes at 37 ℃, add 2.0ml of reagent III to stop the reaction and cool in ice water.
4.Pipette successively the following reagents into a new test tube.
|
0.08 ml |
The terminated solution of step 3 |
|
0.72 ml |
Distilled water |
|
0.4 ml |
α-Naphthol solution (Reagent IV) |
|
0.4 ml |
Alkaline solution (Reagent V) |
|
0.4 ml |
Diacetyl solution (Reagent VI) |
5.Allow to stand for about 1 h at 25°C and dilute by adding 2 ml of distilled water.
6.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).
∆A = As - 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
Suzuki, M. and Yoshida, M., Clin. Chim. Acta, 143, 147– 155 (1984).
