Sarcosine oxidase (SOD-EP)

Enzymes for Clinical Chemistry

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

Origin	recombinant E. coli
Systematic name	Sarcosine : oxygen oxidoreductase (demethylating)
EC Number	1.5.3.1
Reaction formula	Sarcosine + H₂O + O₂ Glycine + Formaldehyde + H₂O₂

SPECIFICATION

Appearance	yellow lyophilizate
Activity	≧20 U/mg
Contaminant	Creatininase <1×10^-2 　U/U%
Stabilizer	sucrose
Storage condition	below -20℃

PROPERTIES

Molecular weight	ca. 49 kDa (gel filtration)
Structure	monomer of 43 kDa (SDS-PAGE) one mole of FAD per mole of enzyme
Isoelectric point	5.3
Michaelis constant	8.5×10^-3M (sarcosine)
pH Optimum	7.5 (Fig. 1)
pH Stability	7.5-9.5 (Fig. 2)
Optimum temperature	50℃ (Fig. 3)
Thermal stability	below 55℃ (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	Zn²⁺,Cu²⁺,Hg²⁺,Ag⁺

APPLICATIONS

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

ASSAY PROCEDURE

Principle

The appearance of quinoneimine dye is measured spectrophotometrically at 495 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of hydrogen peroxide per min at 37℃ and pH 7.7 under the conditions described below.

Reagents

Tris-HCl buffer, 125 mM; pH 7.7: dissolve 15.1 g of Tris(hydroxymethyl)aminomethane in 900 ml of distilled water, adjust to pH 7.7 with 5 N HCl and dilute with distilled water to 1000 ml.
Sarcosine solution, 0.2 M: dissolve 1.78 g of sarcosine in 80 ml of Tris-HCl buffer (Reagent A) containing 0.125% of Triton X-100 and 2.5 mM KCl, adjust to pH 7.7 with 1 N NaOH and dilute with distilled water to 100 ml.
Phenol solution, 0.1%: 100 mg of phenol/100 ml of distilled water.
4-Aminoantipyrine (4-AA) solution, 0.2%: 200 mg of 4-AA/100 ml of distilled water.
Peroxidase (POD) solution, 80 U/ml; 4 mg of POD (200 guaiacol U/mg)/10 ml of distilled water.
Sodium dodecyl sulfate (SDS) solution, 0.3%: 1.5 g of SDS/500 ml of distilled water.
Enzyme dilution buffer: 20 mm Tris-HCl buffer, pH 7.7, containg 1.0 mm KCl and 0.2% bovine serum albumin (BSA).

Sample: dissolve the lyophilized enzyme to a volume activity of 0.06–0.09 U/ml in ice-cold enzyme dilution buffer (Reagent G) immediately before measurement.

Procedure

Prepare the following reaction mixture immediately before use and store on ice in a brownish bottle.

50 ml Sarcosine solution (Reagent B)

20 ml Phenol solution (Reagent C)

10 ml 4-AA solution (Reagent D)

10 ml POD solution (Reagent E)

10 ml Distilled water
Pipette 0.95 ml of the reaction mixture into a cuvette (light path: 1 cm).
Equilibrate at 37℃ for about 5 min.
Add 0.05 ml of sample and incubate for 10 min at 37℃.
Add 2.0 ml of SDS solution (Reagent F) to stop the reaction.
Read the absorbance at 495 nm in a cuvette (light path: 1 cm) (A_S).
The blank solution is prepared by adding enzyme dilution buffer (Reagent G) instead of sample (A₀).