Glucose Dehydrogenase

Glucose Dehydrogenase (GDH) (FAD) is an enzyme that catalyzes the oxidation of glucose using flavin adenine dinucleotide (FAD) as a cofactor, generating gluconolactone and reducing FADH2.

Preparation and specification

Properties

Assay principle

D-Glucose + PMS  ⇒ D-Glucono-1,5-lactone +PMS (red)

PMS (red) +DCIP  ⇒  PMS + DCIP (red)

The assay is based on the increase in absorbance at 600 nm as the formation of DCIP in the forward reactions.

Unit definition

One unit (U) is defined as the amount of enzyme which generates 1 μmol of DCIP per minute at 37 °C under the conditions described below.

Reagents preparation

Reagent I: 0.1 M potassium phosphate buffer, pH 7.0.

Reagent II: 2 M D-glucose solution.

Reagent III: 1.8 mM DCIP solution (be prepared when using).

Reagent IV: 30 mM PMS solution.

Enzyme dilution buffer: 0.1 M potassium phosphate buffer pH 6.0, contains 0.1% BSA.

Sample: The enzyme was diluted to 0.1-0.9 U/mL with enzyme dilution buffer.

Procedure

1. Add 2.05 mL reagent I, 0.6 mL reagent II and 0.15 mL reagent III to the 3 mL cuvette at 37 ℃ for about 5 minutes;

2. Add 0.1 mL reagent IV to the 3mL cuvette and mix;

3. Add 0.1 mL enzyme solution to the 3 mL cuvette and mix;

4. Record the ΔAs at 600 nm in 1 minute in a spectrophotometer thermostated at 37 °C;

5. Replace the enzyme solution with enzyme dilution buffer, and record the change of blank

absorbance (∆Ab) in the same steps;

∆A = ∆As – ∆Ab

Calculation

This enzyme is useful for clinical analysis of D-glucose determination in blood glucose meter.