Creatinase (C2-EP)

Enzymes for Clinical Chemistry

CD : 60144

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

Origin	recombinant E. coli
Systematic name	Creatine amidinohydrolase
EC Number	3.5.3.3
Reaction formula	Creatine + H₂O →→→ Sarcosine + Urea

SPECIFICATION

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

PROPERTIES

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⁺

APPLICATIONS

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).

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

Calculation

Activity can be calculated by using the following formula:

EXPERIMENTAL DATA

Line-up

FAQ

What precautions should be taken when handling the product after opening?: To avoid moisture absorption of the enzyme powder, please allow it to return to room temperature in a sealed state before use. (Recommended conditions: around 25°C, humidity below 55%)

How should C2-EP be stored?: Please store at or below -20°C.

What stabilizers are used in C2-EP?: Sucrose is used as a stabilizer.

How thermostable is C2-EP?: It is stable in liquid form below 50°C (Fig.4) and remains stable at 37°C for at least three weeks (Fig.5). In powder form, it remains stable at 30°C

What are the optimal pH and temperature for C2-EP?: The optimal pH is 7.0–9.0, and the optimal temperature is 45°C.

What is the molecular weight of C2-EP?: The molecular weight is approximately 80 kDa (gel filtration), and it exhibits a dimeric structure composed of subunits of 48 kDa each (SDS-PAGE).