Creatinase (C2-AT) | キッコーマンバイオケミファ

Creatinase (C2-AT) クレアチナーゼ(C2-AT)

臨床診断用酵素

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。

由来	recombinant E. coli
系統名	Creatine amidinohydrolase
EC 番号	3.5.3.3
反応式	Creatine + H2O →→→ Sarcosine + Urea

仕様

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

特性

Molecular weight	ca. 80 kDa (gel filtration)
Structure	2 subunits of 48 kDa (SDS-PAGE)
Michaelis constant	1.2×10^-2M (creatine)
pH Optimum	7.0–9.0 (Fig. 1)
pH Stability	4.0–11.0 (Fig. 2)
Optimum temperature	45℃ (Fig. 3)
Thermal stability	below 53℃ (Fig. 4)
Stability (liquid form)	stable at 37℃ for at least two weeks (Fig. 5)
Stability (powder form)	stable at 30℃ for at least one month (Fig. 6)
Inhibitors	Hg²⁺

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 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

Pipette the following reagents into a test tube.

0.1 ml Potassium phosphate buffer (Reagent A)

0.8 ml Creatine 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 DMAB solution (Reagent C) and allow to stand for about 30 min at 25℃.
Read the absorbance at 435 nm in a cuvette (light path: 1 cm) (A_S).
The blank solution is prepared by reversing the sequence of addition of sample and DMAB solution (Reagent C) (A₀).