Sarcosine oxidase (SOD-EP) ザルコシンオキシダーゼ (SOD-EP)

  • 臨床診断用酵素
SOD-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

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
Contaminant Creatininase <1×10-2 　U/U%
Stabilizer sucrose
Storage condition below -20℃

特性

Molecular weight ca. 49 kDa (gel filtration)
Structure monomer of 43 kDa (SDS-PAGE)
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+

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

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

Calculation

Activity can be calculated by using the following formula:

実験データ

Line-up

参考文献

TOP