Fructosyl-amino Acid Oxidase (FAOD-E) 60273 フルクトシルアミノ酸オキシダーゼ(FAOD-E)

  • 臨床診断用酵素
Fructosyl-amino Acid Oxidaseフルクトシルアミノ酸オキシダーゼ

臨床検査において、フルクトシル-L-アミノ酸の測定に利用されます。

由来 recombinant E. coli
系統名

Fructosyl-L-amino acid : oxygen oxidoreductase
EC 番号 1.5.3
反応式

Fructosyl-L-amino acid + H2O + O2→→→ L-Amino acid + Glucosone + H2O2

仕様

Appearance yellow lyophilizate
Activity ≧4.0 U/mg
Stabilizer trehalose
Storage condition below -20℃ protected from light

特性

Molecular weight ca. 45 kDa (gel filtration)
Structure monomer of 50 kDa (SDS-PAGE)
Michaelis constant 2.2×10-4M (Nε-fructosyl-L-lysine)
pH Optimum 8.0–8.5
pH Stability 6.0–8.5
Optimum temperature 35–40℃
Thermal stability below 30℃
Stability (liquid form) stable at 25℃ for at least two weeks
Stability (powder form) stable at 37℃ for at least three weeks
Inhibitors Ag+，Cu2+
Specificity Nε-fructosyl-L-lysine (100), fructosyl-L-valine (65)
fructosyl-glycine (30)

アプリケーション

The enzyme is useful for the determination of fructosyl-L-amino acid.

分析手順

Principle

The appearance of quinoneimine dye is measured spectrophotometrically at 555 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which produces 1 μmol of hydrogen peroxide per min at 30℃ and pH 8.0 under the conditions described below.

Reagents

  1. Potassium phosphate buffer, 0.1 M; pH 8.0: mix 0.1 M KH2PO4 solution and 0.1 M K2HPO4 solution to make a pH 8.0 solution.
  2. TOOS solution, 0.5%: 125 mg of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)/25 ml of distilled water.
  3. Peroxidase (POD)-4-aminoantipyrine (4-AA) solution: dissolve 5 mg of POD (200 guaiacol U/mg) in 800 ml of potassium phosphate buffer (Reagent A), then add 100 mg of 4-AA and dilute with potassium phosphate buffer (Reagent A) to 1000 ml.
  4. Fructosyl-L-valine solution, 150 mM: 834 mg of fructosyl-l-valine/20 ml of distilled water
  5. Enzyme dilution buffer: 10 mM potassium phosphate buffer, pH 8.0, containing 0.06% n-octhyl-β-D-thioglucoside.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.05–0.18 U/ml in ice-cold enzyme dilution buffer (Reagent E) immediately before measurement.

Procedure

  1. Pipette the following reagents into a cuvette (light path: 1 cm).
    0.1 ml TOOS solution (Reagent B)
    2.7 ml POD-4-AA solution (Reagent C)
    0.1 ml sample
  2. Equilibrate at 30℃ for about 5 min.
  3. Add 0.1 ml of fructosyl-l-valine solution (Reagent D) and mix.
  4. Record the increase of absorbance at 555 nm in a spectrophotometer thermostated at 30℃, and calculate the ΔA
    per min using the linear portion of the curve (ΔAS).
    The blank solution is prepared by adding distilled water instead of fructosyl-l-valine solution (Reagent D) (ΔA0).

Calculation

Activity can be calculated by using the following formula:

39.2 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm2/μmol)
1/2 : Factor based on the fact that 1 mol of hydrogen peroxide produces 1/2 mol of quinoneimine dye
df : Dilution factor
C : Content of fructosyl-amino acid oxidase preparation in sample (mg/ml)

実験データ

FAQ

FAOD-Eの主な用途は何ですか？
FAOD-Eの分子量を教えてください。
FAOD-Eの至適pHと至適温度について教えてください。
FAOD-Eの熱安定性について教えてください。
FAOD-Eの安定化剤は何を使用していますか？
FAOD-Eの基質特異性について教えてください。
FAOD-Eの活性測定方法を教えてください。
FAOD-Eの保存方法を教えてください。
開封後の取扱い方法に注意事項はありますか？

参考文献



お問合せ / サンプル請求

TOP