Fructosyl-Peptide Oxidase

Reagent I: 0.1 M potassium phosphate, pH 8.0.

Reagent II: 1 kU/mL POD.

Reagent III: 50 mM TOOS solution.

Reagent IV: 50 mM 4-AA solution.

Reagent V: 200 mM fructosyl-valine solution.

Enzyme diluent: 20 mM Tris-HCl，pH 8.0.

Sample: Dilute the sample to 0.2-0.5 U/mL by Enzyme diluent.

Reaction mixture:

Reagent I            10 mL

Reagent II           0.1 mL

Reagent III          1 mL

Reagent IV          1 mL

Reagent V           10 mL

Pure water         Set to 100mL

PROCEDURE

1. Add 0.98 mL reaction mixture to the cuvette.
2. Preincubate the reaction mixture at 37 ℃ for 5 min.

3. Add 0.02 ml of the enzyme solution to the reaction mixture.

4. Record the ΔAs at 555 nm in 1 minute in a spectrophotometer Thermo stated at 37 ℃. At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.

                           ∆A = ∆As – ∆Ab

Vt: Total volume (1.0 mL)

Vs: Enzyme volume (0.02 mL)

t: reaction time (1 min)

df: dilution factor

C: Enzyme concentration (mg/mL)

1.0: Light path length (cm)

1/2: 1mol H2O2 will react to 1/2 mol

Quinoneimine dye

39.2: Millimolar extinction coefficient of

quinone imine dye under 555nm (cm2/μmol)