Creatininase (C1-E)

Enzymes for Clinical Chemistry

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

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

Pipette the following reagents into a test tube.

0.1 ml Potassium phosphate buffer (Reagent A)

0.8 ml Creatinine solution (Reagent B)
Equilibrate at 37℃ for about 5 min.
Add 0.1 ml of sample and incubate for 10 min at 37℃.
Add 2.0 ml of sodium carbonate solution (Reagent C) to stop the reaction and cool in ice water.
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)
Allow to stand for about 1 h at 25℃ and dilute by adding 2.5 ml of distilled water.
Read the absorbance at 525 nm in a cuvette (light path: 1 cm) (A_S).
The blank solution is prepared by reversing the sequence of addition of sample and sodium carbonate solution (Reagent C) (A₀).

0.1 ml	Potassium phosphate buffer	(Reagent A)
0.8 ml	Creatinine solution	(Reagent B)

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)