规格

特性

适用类型

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.)

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

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 37℃ for about 5 min.
3. Add 0.1 ml of fructosyl-glycine solution (Reagent D) and mix.
4. 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).

Calculation

Activity can be calculated by using the following formula:

EXPERIMENTAL DATA

Line-up

• Fructosyl-peptide Oxidase (FPOX-CE)
• Fructosyl-peptide Oxidase (FPOX-CET)

参考文献

Hirokawa K, Shimoji K, Kajiyama N. (2005)
An enzymatic method for the determination of hemoglobinA1C.
Biotechnology Letters, 27(14), 963-968.

Hirokawa K, Ichiyanagi A, Kajiyama N. (2008)
Enhancement of thermostability of fungal deglycating enzymes by directed evolution.
Applied Mcrobiology and Biotechnology, 78(5), 775-781.

Ichiyanagi A, Hirokawa K, Gomi K, Nakatsu T, Kato H, Kajiyama N. (2013)
Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 69(2), 130-133.