Sucrose/Fructose/Glucose Video Protocol with K-SUFRG

Sucrose/Fructose/Glucose Video Protocol with K-SUFRG

January 12, 2020 3 By Jose Scott


Megazyme provides an extensive range of assay kits for use in various assay formats including auto-analyzer, microplate and manual spectrophotometer. This sucrose/D-fructose/D-glucose assay kit can be used to accurately measure
sucrose, D-fructose and D-glucose in various sample types
throughout industries such as food, dairy, and beverages.
This video tutorial will demonstrate the use of the kit with a fruit juice sample
using the manual spectrophotometer format. The kit contains sufficient reagents for either
100 or 200 manual assays and is supplied with a detailed data booklet. The assay is specific for the measurement
of sucrose, D-fructose, and D-glucose. The principle of the enzymatic reactions involved
in the measurement of sucrose, D-fructose and D-glucose are shown in this figure. Sucrose is hydrolysed to D-fructose and D-glucose. The amount of NADPH formed in these reactions
is stoichiometric with the amount of D-glucose and D-fructose in the cuvette. It is the NADPH which is measured by the increase in absorbance at 340 nm. Prior to sample analysis, the kit components
should be prepared as described in the kit data booklet, and once prepared, they are ready for use in the manual assay procedure. All of the kit components except Bottles 2 and 6 are used as supplied. The contents of Bottle 2 are dissolved in
22 mL of distilled water. The bottle is capped and the contents are
mixed thoroughly to ensure complete dissolution. The contents of Bottle 6 are dissolved in 20 mL of distilled water. For longer-term storage, divide into aliquots
of approximately 5 mL and store at below -10 degrees Celsius. To prepare the sucrose standard,
accurately weigh 0.5 g of crystalline sucrose into a 1 L volumetric flask
and dissolve in distilled water. Adjust to the mark with distilled water.
Store in appropriately-sized aliquots below -10 degrees Celsius.
Keep cool during use. Follow the manual assay procedure as described
in the data booklet. This assay requires each sample to be applied
to two assays: a sucrose assay for measurement of sucrose,
and a D-glucose/D-fructose assay for measurement of D-glucose and D-fructose.
A blank reaction and a standard reaction must be performed with each batch of samples for
both assays. In this demonstration, we are using one sample
tube per assay (1 Suc and 1 G/F), 1 blank tube (BL Suc and BL G/F)
and 1 standard tube (ST Suc and ST G/F) for each assay. Pre-warm Bottle 6 (beta-fructosidase) and
pipette 0.2 mL into all sucrose (Suc) assay tubes. Pipette 0.1 mL of sample into sample assay tubes
(1 Suc and 1 G/F). Pipette 0.1 mL of sucrose standard to the sucrose standard tube (ST Suc). Pipette 0.1 mL of Bottle 5 (glucose/fructose standard) into the glucose/fructose standard
assay tube (ST G/F). When all of the components have been added,
mix the tube contents thoroughly and incubate the tubes between 25 and 27 degrees Celsius
for approx. 5 minutes. Pipette the required volume of distilled water
into all assay tubes. Pipette 0.1 mL of Bottle 1 (buffer) into all
assay tubes. Pipette 0.1 mL of Bottle 2 (NADP+ and ATP)
into all assay tubes. When all of the components have been added,
mix the tube contents thoroughly and incubate the tubes between 25 and 37 degrees Celsius
for approx. 3 minutes. For the purpose of this Sucrose/D-Fructose/D-Glucose
assay kit, the MegaQuant Wave Spectrophotometer is used
in absorbance mode. After 3 minutes, record the first absorbance
reading (A1) at 340 nm for all of the assay tubes. In this demonstration, we are using the MegaQuant
Wave spectrophotometer set to read at 340 nm. Alternatively, a recording spectrophotometer with a 1 cm path length cuvettes can be used. Swirl to mix the contents of Bottle 3 (hexokinase/G6P
dehydrogenase) prior to dispensing. After recording the A1 absorbance value, pipette
0.02 mL of Bottle 3 into all assay tubes. Mix the tube contents thoroughly
and incubate the tubes between 25 and 37 degrees Celsius for approx. 5 minutes. After 5 minutes, record the second absorbance
reading, A2, at 340 nm for all of the assay tubes. This absorbance reading completes the sucrose
assay. The D-glucose/D-fructose assay requires further
analysis. Swirl to mix the contents of Bottle 4 (phosphoglucose
isomerase) prior to dispensing. Pipette 0.02 mL of Bottle 4 into all Glucose/Fructose
(G/F) assay tubes. Mix the tube contents thoroughly
and incubate the tubes between 25 and 37 degrees Celsius for approx. 10 minutes.
After 10 minutes, record the third absorbance reading (A3) at 340 nm for all of the Glucose/Fructose
assay tubes. This absorbance reading completes the
D-Glucose/D-Fructose assay. The relevant absorbance readings A1, A2 and
A3 of the sample and blank reactions are used to calculate the sucrose, D-glucose
and D-fructose concentrations in the original sample. When performing this test on the MegaQuant
Wave spectrophotometer, the absorbance mode feature must be used.
The results will be printed automatically via the onboard printer
or the data can be exported to a computer using the SF Capture software.
Please see our MegaQuant Wave video for further details. In absorbance mode, the results output are
raw absorbance values for both blank and samples. Therefore, the calculation of sucrose, D-glucose
and D-fructose content can be performed manually as described in
the Calculations section of the kit booklet. Megazyme has also developed specific Excel-based
MegaCalc applications for each Megazyme kit to allow quick and easy results analysis.
Results can be analysed using the MegaCalc application specific to this
Sucrose/D-Fructose/D-Glucose kit which is available to download free of charge
from the Megazyme website. The MegaCalc spreadsheet provides full instructions
for use. Open the MegaCalc worksheet and input the
following: sample details;
absorbance readings for the blanks; for each sample input the sample identifier
and the absorbance values for the samples. Alter the sample volume if a volume other
than the default 0.1 mL is used. If dilution of the sample has been performed,
then input the dilution factor used. If no further dilution was performed, the
dilution factor is 1. When all of the data has been entered, the
concentration of sucrose, D-glucose and D-fructose in the sample is automatically calculated
and given as grams per liter in the original sample. For solid samples, input the concentration
of the original sample extract in grams per liter. The concentration of sucrose, D-glucose and D-fructose in the solid sample is then automatically
calculated and provided as grams per 100g in the original sample.