Assay Procedure for D-Fructose Dehydrogenase

Principle

Prussian blue : KFeⅢ[FeⅡ(CN)6]
The appearance of prussian blue formed by chelate reaction is measured at 660nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of D-fructose (the formation of two micromoles of prussian blue) per minute under the conditions described below.

Method

Reagents

 

A. McⅠlvaine buffer, pH 4.5 :Prepare by mixing of 0.1M citric acid and 0.2M disodium phosphate, at 25℃
B. D-Fructose solution :1.0M (1.80g D-fructose (MW=180.16)/10ml McⅠlvaine buffer (A) contg. 0.1% Triton X-100)
C. Potassium ferricyanide solution :0.1M (0.33g potassium ferricyanide (MW=329.25)/10ml McⅠlvaine buffer (A) contg. 0.1% Triton X-100)
D. Ferric sulfate-SDS solution :5.0g Fe2(SO4)3・H2O, 3.0g SDS (sodium dodecyl sulfate), 95ml 85% phosphoric acid/1,000ml of H2O
E. Enzyme diluent :McⅠlvaine buffer (A) contg. 0.1% Triton X-100 and 0.05% BSA

Procedure

  1. Pipette 0.7ml of Reagent E, 0.1ml of Reagent B and 0.1ml of the enzyme solution* into a test tube and equilibrate at 37℃ for about 5 minutes.
Concentration in assay mixture
McⅠlvaine buffer ×1
Triton X-100 0.1%
D-Fructose 0.1M
Potassium ferricyanide 10mM
  1. Add 0.1ml of Raegent C and mix.
  2. After exactly 5 minutes at 37℃, add 0.5ml of Reagent D to stop the reaction, and then incubate at 37℃ for further 20 minutes.
  3. Add 3.5ml of distilled water and measure the optical density at 660nm against water (OD test).

At the same time, prepare the blank by using the same method as the test except that Reagent E (0.1ml) is used instead of the Reagent B (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent and dilute to 1.0-3.0U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula:

 

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (5.0ml)
Vs :Sample volume (0.1ml)
2.0 : Millimolar extinction coefficient of prussian blue under the assay conditions (F/micromole)
2 :Factor based on the fact that oxidation of one mole of D-fructose produces two moles of prussian blue
t :Reaction time (5 minutes)
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
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