D-Fructose/D-Glucose Assay Procedure (K-FRUGL)
Megazyme provides an extensive range of assay kits for use in various assay formats, including autoanaylser, microplate and manual spectrophotometer This D-Fructose and D-Glucose Assay kit can be used to accurately measure D-Fructose and or D-Glucose in various sample types, throughout industries such as food and beverages. This video tutorial will demonstrate the use of the assay kit with a wine sample, using the manual spectrophotometer format. It is important to measure D-Fructose and D-Glucose in wine, as they are the major nutrients for alcoholic fermentation. The level of residual sugar dictates the sweetness of wine and contributes to the flavour and mouthfeel. This kit contains sufficient reagents for either 110 or 220 manual assays and is supplied with a detailed data booklet. These assays are specific for the measurement of D-Glucose and D-Fructose. The principle of the enzymatic reactions involved in the measurement of D-Glucose and D-Fructose is shown in this figure. The enzyme hexokinase simultaneously phosphorylates D-Glucose to glucose-6-phosphate plus ADP and D-Fructose to fructose-6-phosphate plus ADP. In the presence of the enzyme glucose-6-phosphate dehydrogenase, glucose-6-phosphate is oxidised by NADP+ to gluconate-6-phosphate with the formation of NADPH. The amount of NADPH formed in this reaction is stoichiometric with the amount of D-Glucose. It is the NADPH which is measured by the increase in absorbance at 340 nm. On completion of the glucose measurement, fructose-6-phosphate is converted to glucose-6-phosphate by the addition of the phosphoglucose isomerase. The glucose-6-phosphate formed reacts in turn with NADP+ forming gluconate-6-phosphate and NADPH, leading to a further rise in absorbance that is stoichiometric with the amount of D-Fructose. 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 bottle 2 are used as supplied. The contents of bottle 2 are dissolved in 12 mL of distilled water. The bottle is capped and the contents are mixed thoroughly to ensure complete dissolution. Follow the manual assay procedure as described in the data booklet. Pipette all assay components except the trigger enzymes in bottles 3 and 4 into each assay tube. A blank reaction and a standard reaction must be performed with each batch of samples. Pipette 2.0 mL of distilled water into all assay tubes. Pipette 0.1 mL of sample to sample assay tubes. Pipette 0.1 mL of distilled water into blank assay tube. Pipette 0.1 mL of bottle 5 kit standard into standard assay tube. Pipette 0.1 mL of solution 1 into all assay tubes. Pipette 0.1 mL of solution 2 into all assay tubes. When all of the components have been added, mix the tube contents thoroughly and incubate the tubes in the 37 °C heating block for approximately 3 minutes. 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 1 cm path length cuvettes can be used. Swirl to mix the contents of bottle 3 prior to dispensing. After recording the A1 absorbance value, pipette 20 μL of the first trigger enzyme hexokinase glucose-6-phosphate dehydrogenase into all assay tubes. Mix the tubes thoroughly and incubate them at 37 °C for 5 minutes to allow the reaction to go to completion. Record the absorbance reading A2 for all of the assay tubes. This absorbance reading completes the measurement of D-Glucose. Swirl to mix the contents of bottle 4 prior to dispensing. After recording the A2 absorbance value, pipette 20 μL of the second trigger enzyme phosphoglucose isomerase into all assay tubes. Mix the tubes thoroughly and incubate them at 37 °C for 10 minutes to allow the reaction to go to completion. Record the absorbance reading A3 for all of the assay tubes. This absorbance reading completes the measurement of D-Fructose. The absorbance readings A1 and A2 of the sample and the blank reactions are used to calculate the D-Glucose concentrations in the original samples. The absorbance readings A2 and A3 of the sample and blank reactions are used to calculate the D-Fructose concentrations in the original samples. When performing this test using the pre-installed protocol on the MegaQuant Wave Spectrophotometer, the results will be automatically calculated and printed 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. If the results output a raw absorbance values for both blank and samples, the calculations of D-Fructose and D-Glucose content can be performed manually as described in the calculation section of the kit booklet. Megazyme has also developed a specific excel based Mega-Calc applications for each Megazyme kit to allow quick and easy results analysis. Results can be analysed using the Mega-Calc application specific to this D-Fructose/D-Glycose assay kit which is available to download free of charge from the Megazyme website. The Mega-Calc spreadsheet provides full instructions for use. Open the Mega-Calc worskeet 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 D-Glucose and D-Fructose in the sample is automatically calculated and given as g/L in the original sample. For solid samples, input the concentration of the original sample extract in g/L. The concentration D-Glucose and D-Fructose in the solid samples is then automatically calculated and provided as grams per 100 gram in the original sample.