Creatinase (C2-AE) | 龟甲万百欧凯米发株式会社

Creatinase (C2-AE) 肌酸酶

临床检测用酶

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

由来	recombinant E. coli
系统名称	Creatine amidinohydrolase
EC编号	3.5.3.3
反应式	Creatine + H2O →→→ Sarcosine + Urea

规格

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 46 kDa (SDS-PAGE)
Michaelis constant	1.3×10^-2M (creatine)
pH Optimum	7.0–9.0 (Fig. 1)
pH Stability	5.0–11.0 (Fig. 2)
Optimum temperature	40℃ (Fig. 3)
Thermal stability	below 45℃ (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⁺

适用类型

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

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).