Creatininase (C1-E)

  • Enzymes for Clinical Chemistry
Creatininase (C1-E)

CD : 60111

The enzyme is useful for the determination of creatinine in clinical analysis.

For recipe information



Img. Ref.:NAR 46: W296–W303 (2018)

Origin recombinant E. coli
Systematic name

Creatinine amidohydrolase
EC Number 3.5.2.10
Reaction formula

Creatinine H2O Creatine

SPECIFICATION

Appearance white lyophilizate
Activity 600-750 U/mg
Contaminants Catalase　≦0.5 U/U ％
Stabilizer sucrose
Storage condition below -20℃

PROPERTIES

Molecular weight ca. 170 kDa (gel filtration)
Structure 6 subunits of 28 kDa (SDS-PAGE)
Isoelectric point 4.8
Michaelis constant 3.4×10-2M (creatinine)
4.3×10-2M ((creatine)
pH Optimum 6.5–7.0 (Fig. 1)
pH Stability 7.0–11.0 (Fig. 2)
Optimum temperature 60–65℃ (Fig. 3)
Thermal stability below 60℃ (Fig. 4)
Stability (liquid form) stable at 37°C for at least two weeks (Fig. 5)
Stability (powder form) stable at 30°C for at least one month (Fig. 6)
Inhibitors Hg2+
Activators Mg2+，Mn2+ 

APPLICATIONS

The enzyme is useful for the determination of creatinine and creatine in clinical analysis.

ASSAY PROCEDURE

Principle

The appearance of creatine is measured spectrophotometrically at 525 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of creatine per min at 37℃ and pH 6.8 under the conditions described below.

Reagents

  • Potassium phosphate buffer, 0.3 M; pH 6.5: mix 0.3 M KH2PO4 solution and 0.3 M K2HPO4 solution to make a pH 6.5 solution.
  • Creatinine solution, 0.1 M: 1.13 g of creatinine/100 ml of distilled water.
  • Sodium carbonate solution, 4%: 4.0 g of Na2CO3 (anhydrous)/100 ml of distilled water.
  • α-Naphthol solution, 2%: 2.0 g of α-naphthol/100 ml of ethanol (99.5%).
  • Alkaline solution, 1.2% NaOH, 3.2% Na2CO3: dissolve 1.2 g of NaOH and 3.2 g of Na2CO3 (anhydrous) in 80 ml of distilled water and dilute with distilled water to 100 ml.
  • Diacetyl solution, 0.05%: 0.05 ml of diacetyl/100 ml of distilled water.
  • Enzyme dilution buffer: dissolve 61 mg of Tris(hydroxymethyl)aminomethane in 80 ml of distilled water, adjust to pH 8.0 with 1 N HCl and dilute with distilled water to 100 ml.

Sample: dissolve the lyophilized enzyme to a volume activity of 2–4 U/ml with ice-cold enzyme dilution buffer (Reagent G) immediately before measurement.

Procedure

  1. Pipette the following reagents into a test tube.
    0.1 ml Potassium phosphate buffer (Reagent A)
    0.8 ml Creatinine solution (Reagent B)
  2. Equilibrate at 37℃ for about 5 min.
  3. Add 0.1 ml of sample and incubate for 10 min at 37℃.
  4. Add 2.0 ml of sodium carbonate solution (Reagent C) to stop the reaction and cool in ice water.
  5. Pipette successively the following reagents into a test tube.
    0.1 ml The terminated solution of step 4
    0.9 ml Distilled water
    0.5 ml α-Naphthol solution (Reagent D)
    0.5 ml Alkaline solution (Reagent E)
    0.5 ml Diacetyl solution (Reagent F)
  6. Allow to stand for about 1 h at 25℃ and dilute by adding 2.5 ml of distilled water.
  7. 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 C) (A0). 

Calculation

Activity can be calculated by using the following formula:

EXPERIMENTAL DATA

Line-up

FAQ

What precautions should be taken when handling the product after opening?
How should C1-E be stored?
What stabilizers are used in C1-E?
How thermostable is C1-E?
What are the optimal pH and temperature for C1-E?
What is the molecular weight of C1-E?
What are the main applications of C1-E?

REFERENCES

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.



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