Creatininase (C1-E) | キッコーマンバイオケミファ

Creatininase (C1-E) クレアチニナーゼ(C1-E)

臨床診断用酵素

臨床検査において、クレアチニンの測定に利用されます。

由来	recombinant E. coli
系統名	Creatinine amidohydrolase
EC 番号	3.5.2.10
反応式	Creatinine H2O Creatine

仕様

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

特性

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⁺

アプリケーション

臨床検査において、クレアチンおよびクレアチニンの測定に利用されます。

分析手順

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)