Sarcosine Oxidase (SOD-TE) | キッコーマンバイオケミファ

Sarcosine Oxidase (SOD-TE) サルコシンオキシダーゼ(SOD-E)

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	Sarcosine : oxygen oxidoreductase (demethylating)
EC 番号	1.5.3.1
反応式	Sarcosine + H2O + O2 →→→ Glycine + Formaldehyde + H2O2

仕様

Appearance	yellow lyophilizate
Activity	≧20 U/mg
Contaminants	Creatininase　<1.0×10^-2 U/U%
Stabilizer	sucrose
Storage condition	below -20℃

特性

Molecular weight	ca. 49 kDa (gel filtration)
Structure	monomer of 43 kDa (SDS-PAGE)
Structure	one mole of FAD per mole of enzyme
Isoelectric point	5.3
Michaelis constant	8.3×10^-3M (sarcosine)
pH Optimum	6.7–9.5 (Fig. 1)
pH Stability	6.5–10.5 (Fig. 2)
Optimum temperature	50℃ (Fig. 3)
Thermal stability	below 55℃ (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	Zn²⁺，Cu²⁺，Hg²⁺，Ag⁺

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 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).