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Tuesday, April 26, 2011

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 oxidises it to a red chromophore product.

Glucose + O2 --------------→ H2O2 + Gluconic Acid [enzyme Glucose oxidase catalyses the reaction ]

H2O2 + O-dianisidine Peroxidase ---------→ Red-coloured product [enzyme peroxidase catalyses the reaction ]


MATERIALS

  • Glucose Oxidase Peroxidase Reagent

Dissolve 25 mg O-dianisidine completely in 1 mL of methanol. Add 49 mL of 0.1 M phosphate buffer (pH 6.5). Then add 5 mg of peroxidase and 5 mg of glucose oxidase to the above prepared O-dianisidine solution.

  •  Standard: Dissolve 100 mg glucose in 100 mL water. Dilute 10 mL of this stock to100 mL to obtain the working standard.

PROCEDURE

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

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

3. Incubate all the tubes at 35°C for 40 minutes.

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

5. Read the colour intensity at 540 nm.

CALCULATION

From the standard graph, calculate the amount of glucose present in the sample preparation, see "Determination of Reducing Sugars by Nelson-Somogyi Method" for calculation.

REFERENCES

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.

3. Internet sources