D-Glucose - UV-Test

 

Principle

 

D-Glucose is posphorylated by Hexokinase (HK) and Adenosin-5'-tri-phosphat (ATP) forming Glucose-6-phosphat (G-6-P) and von Adenosin-5'-diphosphat (ADP).

 

D Glucose + ATP -> G-6-P + ADP

 

In presence of Glucose-6-phosphat-Dehydrogenase (G6P-DH) G-6-P will be oxidized by NADP forming Gluconat-6-phosphate. Thus, reduced NADPH is produced.

 

G-6-P + NADP+ -> Gluconat-6-phosphate + NADPH + H⁺

 

The amount of NADPH formed is prop. to the glucose-concentration.

NADPH is determined photometrically at 334, 340 or 365 nm.

 

Reagents(Roche Test-Kit)

 

1.

3 vials with 7,2 g powder mix from:

   triethanol amine buffer, pH 7,6,

   NADP 110 mg,

   ATP 260 mg,

   magnesium sulfate and

   stabilizer.

 

2.

3 vials with 1,1 ml enzyme suspension from

   Hexokinase, about 320 U and

   Glucose 6-phosphat-Dehydrogenase, 160 U.

 

Prep. Of reagents:

1. Vial 1 + 45 ml water.

2. Vial 2 as is.

 

Assay:

 

Cuvette (glass) :            1 cm

Temperature:                 20-25°C

Volume:                          3 ml

Sample:                          4-100 µg D-Glucose / cuvette

 

Mix solution 1 (1ml), sample (0.1 ml), Aqua dest. (1.9 ml) and solution 2 (20 µl), wait about 10-15 minutes, measure absorbance.