Enzymatic Method for Carbohydrate Determination


This video will demonstrate how to perform
carbohydrate analysis using enzymatic method. Here is the equipment you will need. Six plastic cuvettes will be provided, three
for the blank and three for the sample. The Enzymatic Kit includes: Solution 1 – which
provides the components such as NADP and ATP for the enzymatic reactions; Suspension 2
– which provides hexokinase and glucose-6 phosphate dehydrogenase; Suspension 3 – which
provides the phosphoglucose isomerase. All of these have to be kept on ice until
use. Weigh 5 ± 0.1 g of apple puree in the beaker. Add ddwater into the beaker and pour it into
the 50 ml volumetric flask. Rinse the beaker and add it into the flask,
then make up to volume. Filter the apple puree solution into a test
tube. Refer to your pre-lab calculation to get an
idea on how much solution you will need. Dilute the apple puree to reach 250 µg sugar
per ml concentration in ddwater in a microfuge tube. Prepare enough volume for the enzymatic assay. Follow the Enzymatic assay guide in your lab
manual to add the appropriate volume of Solution 1, sample solution and ddwater into the plastic
cuvette. Remember to use only half of the volume. Remember to prepare your sample blanks. Use the pipetman to mix the solution by pipetting
up and down, then switch to a new pipette tip for the next cuvette. Incubate for 3 minutes at room temperature
then read the absorbance at 340 nm using the Shimadzu spectrophotometer or TECAN spectrophotometer. Add the proper volume of suspension 2 directly
into the cuvette and mix by pipetting up and down. Incubate for 10 minutes at room temperature
then read the absorbance. Finally, add the proper amount of suspension
3 directly into the cuvette and mix by pipetting up and down. Incubate for another 10 minutes before reading
the absorbance. Calculate the amount of glucose and fructose
in the puree sample using the information in the Enzymatic assay guide. Discard the solution down the sink and dispose
the plastic cuvettes into the garbage bin.

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