Fructosyl-peptide Oxidase (FPOX-CET)

Enzymes for Clinical Chemistry

The enzyme is useful for the determination of fructosyl-peptide and fructosyl-L-amino acid. Also it is useful for the determination of HbA1c by using protease together. (HbA1c is used as a test makar to diagnose diabetes. HbA1c can also be quantified by measuring the fructosyl peptide or fructosyl-L-amino acid excised？ by the protease from HbA1c.)

Origin	recombinant E. coli
Systematic name	Fructosyl-peptide : oxygen oxidoreductase
EC Number	1.5.3
Reaction formula	Fructosyl-L-amino acid + H2O + O2 →→→ Peptide + Glucosone + H2O2

SPECIFICATION

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

PROPERTIES

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)

APPLICATIONS

ASSAY PROCEDURE

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).