Creatinase (C2-EP) クレアチナーゼ(C2-EP)

  • 臨床診断用酵素
C2-EP

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 1.1×10-2M (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+

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

  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:

実験データ

Line-up

参考文献

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