Methodology for Carbohydrates

Determination of glucose by glucose oxidase method


Glucose is a widely distributed simple sugar with an active aldehyde group. Estimation of glucose by glucose oxidase gives the true glucose concentration eliminating the interference by other reducing sugars.




Principle

Glucose oxidase catalyses the oxidation of alpha-D-glucose to D-glucono-1, 5 lactone (gluconic acid) with the formation of hydrogen peroxide. The oxygen liberated from hydrogen peroxide by peroxidase reacts with the O-dianisidine and oxidizes it to a red chromophore product.


Glucose

Glucose + O2

H2O2 + Gluconic Acid

Oxidase



H2O2 + O-dianisidine       
Peroxidase
Red-colored product






Materials


» Glucose Oxidase Peroxidase Reagent:
» Dissolve 25mg O-dianisidine completely in 1mL of methanol. Add 49mL of 0.1M phosphate buffer (pH 6.5). then add 5mg of peroxidase and 5mg of glucose oxidase to the above prepared O-dianisidine solution.
» Standard: Dissolve 100mg glucose in 100mL water. Dilute 10mL of this stock to 100mL to obtain the working standard.


Procedure

1.

To 0.5mL of deprotinised plant extract (deproteinization is not necessary in samples with very low protein content) add 0.5mL distilled water and 1mL glucose oxidase- peroxidase reagent.

2.

Into a series of test tube pipette out 0 (blank). 0.2, 0.4, 0.6, 0.8 and 1mL of working standard glucose solution and make up the volume to 1.0mL with distilled water. Then add 1mL of glucose oxidase-peroxidase reagent.

3.

Incubate all the tube at 35°C for 40 minutes.

4.

Terminate the reaction by the addition of 2mL of 6 N-HCl.

5.

Read the color intensity at 540nm.



Calculation

From the standard graph, calculate the amount of glucose present in the sample preparation.



Reading

1. Malik, C P and Singh, M B (1980) Plant Enzymology and Histoenzymology Kalyani Publishers New Delhi p 278.
2. Krishnaveni, S, Theymoli Balasubramanian and Sadasivam, S (1984) Food Chem 15 229.