Fructosyl-peptide Oxidase (FPOX-CET) | キッコーマンバイオケミファ

Fructosyl-peptide Oxidase (FPOX-CET) フルクトシルペプチドオキシダーゼ(FPOX-CET)

臨床診断用酵素

臨床検査において、フルクトシル-L-アミノ酸およびフルクトシルペプチドの測定に利用されます。また、プロテアーゼと併用することでHbA1cの測定に利用されます。(HbA1cは糖尿病の診断に検査指標として利用されています。プロテアーゼによって HbA1c から切り出されたフルクトシルペプチドまたはフルクトシル-L-アミノ酸をFPOXで測定することによって、HbA1cを定量化することが可能です。)

由来	recombinant E. coli
系統名	Fructosyl-peptide : oxygen oxidoreductase
EC 番号	1.5.3
反応式	Fructosyl-L-amino acid + H2O + O2 →→→ Peptide + Glucosone + H2O2

仕様

Appearance	yellow lyophilizate
Activity	≧5.0 U/mg
Contaminants	Catalase　≦1.0 U/U％
Stabilizer	sodium glutamate, EDTA
Storage condition	below -20℃

特性

Molecular weight	ca. 60 kDa (gel filtration)
Structure	monomer of 52 kDa (SDS-PAGE)
Michaelis constant	1.5×10^-3M (fructosyl-valyl-histidine) 5.0×10^-3M (fructosyl-glysine) 9.0×10^-3M (N^ε-fructosyl-lysine)
pH Optimum	7.5–8.5
pH Stability	5.5–9.5
Optimum temperature	37–45℃
Thermal stability	below 45℃
Stability (powder form)	stable at -20℃ for at least 12 months
Specificity	fructosyl-valyl-histidine (100), fructosyl-glycine (150)
Specificity	N^ε-fructosyl-L-lysine (68.6)

アプリケーション

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 37℃ and pH 8.0 under the conditions described below.

Reagents

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.
TOOS solution, 15 mM: 0.50 g of TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine)/100 ml of distilled water.
Peroxidase (POD)-4-aminoantipyrine (4-AA) solution: dissolve 5 mg of POD preparation (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.
Fructosyl-glycine solution, 600 mM: 1.43 g of fructosyl-glycine/10 ml of distilled water.
Enzyme dilution buffer: 10 mM potassium phosphate buffer, pH 8.0, containing 0.15% bovine serum albumin (BSA).

Sample: dissolve the lyophilized enzyme to a volume activity of 0.2–0.5 U/ml in ice-cold enzyme dilution buffer (Reagent E) immediately before measurement.

Procedure

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
Equilibrate at 37℃ for about 5 min.
Add 0.1 ml of fructosyl-glycine solution (Reagent D) and mix.
Record the increase of absorbance at 555 nm in a spectrophotometer thermostated at 37℃ , and calculate the ΔA per min using the linear portion of the curve (ΔA_S).
The blank solution is prepared by adding distilled water instead of fructosyl-glycine solution (Reagent D) (ΔA₀).