Creatinase (C2-EP)

  • Enzymes for Clinical Chemistry
C2-EP

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

Origin recombinant E. coli
Systematic name

Creatine amidinohydrolase
EC Number 3.5.3.3
Reaction formula

Creatine + H2O →→→ Sarcosine + Urea

SPECIFICATION

Appearance white to light yellow lyophilizate
Activity ≧9 U/mg
Stabilizer sucrose
Storage condition below -20℃

PROPERTIES

Molecular weight ca. 80 kDa (gel filtration)
Structure 2 subunits of 48 kDa (SDS-PAGE)
Michaelis constant 1.1×10-2M (creatine)
pH Optimum 7.0–9.0 (Fig. 1)
pH Stability 5.0-9.0 (Fig. 2)
Optimum temperature 45℃ (Fig. 3)
Thermal stability below 50℃ (Fig. 4)
Stability (liquid form) stable at 37°C for at least three weeks (Fig. 5)
Stability (powder form) stable at 30°C for at least three weeks (Fig. 6)
Inhibitors Hg2+

APPLICATIONS

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

  1. Pipette the following reagents into a test tube.
    0.1 ml Potassium phosphate buffer (Reagent A)
    0.8 ml Creatine 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 DMAB solution (Reagent C) and allow to stand for about 30 min at 25℃.
  5. 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

REFERENCES

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