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| Section: Biotechnology Methods » Enzymology |
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| Construction of the Maltose Calibration Curve |
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Principle
Maltose is a reducing disaccharide. Maltose reduces the alkaline solution of
3.5 dinitro salicylic acid (DNS), which is pale yellow, into an orange-red complex
of 3-amino –5-nitro salicylic acid. The optical density is measured at 540 nm.
The intensity of the color depends on the concentration of maltose.
Reagents
- 3,5–dinitrosalicylic acid (DNS):
Dissolve 10 gms of DNS in 200 mL of 2N NaOH. To this, add 500 mL of
distilled H2O and 300 mL of sodium potassium tartrate and make up the
volume to 100 mL with distilled H2O.
- Standard maltose solution: 1 mg/mL in distilled H2O.
- Distilled H2O.
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Procedure
Pipette out standard maltose solution ranging from 0.0 to 20 mL into test tubes
and make up the volume to 2.0 mL with distilled water. The first tube, with
2.0 mL of distilled water, serves as the blank.
Add 1 mL of alkaline DNS-reagent to each tube. Keep all the tubes in
boiling water bath for exactly 5 minutes and cool to room temperature. Add
4 mL of distilled water to each tubes and read the OD at 540 nm.
Plot the graph with maltose along the x-axis and the optical density along
the y-axis.
Result: The maltose calibration curve is constructed as a straight line passing
through the origin. |
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