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Experimental Procedure – Practical Work 2

Determination of Reducing Sugars by the Dinitrosalicylic Acid (DNS) Method

In this practical class, we will use the supernatants obtained in Practical Class 1 and quantify glucose using an alternative method to HPLC - the dinitrosalicylic acid (DNS) method.

The method used was described by Miller (1959). This method is based on the formation of a brown-orange complex by the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid by reducing sugars. The concentration of this colored complex is proportional to the concentration of reducing sugars in the sample. From known values of glucose concentration, it is possible to draw a calibration curve, such as the one shown in the following figure, illustrating the absorbance (OD at 540 nm) as a function of glucose concentration in reducing sugar equivalents.

Calibration Curve for Reducing Sugars in Glucose Equivalents by the DNS Method

The equation of the line obtained by linear regression is as follows:


[ \text{[Reducing Sugars in Glucose Equivalents, g/L]} = 1.89 \times \text{OD} (540 \text{nm}) + 0.002 ]

Preparation of DNS Reagent:

• 5 g of 3,5-dinitrosalicylic acid was added to 100 mL of sodium hydroxide solution (2N) heated to 80°C. To 250 mL of hot distilled water (80°C), 150 g of sodium potassium tartrate was added. The two solutions were mixed to make a final volume of 500 mL. This solution is stored at room temperature protected from light.

Experimental Protocol for Practical Class 2:

1. Each group should prepare 4 test tubes (corresponding to the 4 incubation times used in Practical Class 1) and add 0.5 mL of the solution to be analyzed (sample obtained in Practical Class 1) and 0.5 mL of the DNS solution, according to the following table:

Group / Bench	Experimental Condition	Samples	Volume of Sample	Volume of DNS
1	S. cerevisiae / aerobic	0	0.5 mL	0.5 mL
2	S. cerevisiae / anaerobic	0	0.5 mL	0.5 mL
3	T. delbrueckii / aerobic	0	0.5 mL	0.5 mL
4	T. delbrueckii / anaerobic	0	0.5 mL	0.5 mL

2. Incubate for 5 minutes in a boiling water bath.

3. Cool under running water (tap).

4. Add 5 mL of cold deionized water to each tube and vortex.

5. Measure absorbance at 540 nm using a spectrophotometer. Before measurement, prepare a blank using 0.5 mL of deionized water + 0.5 mL of DNS.

6. Calculate the concentration of reducing sugars using the formula provided in the previous image (this calibration curve was obtained using standard glucose solutions of known concentrations).

Reference:

• Miller, G. L (1959) “Use of DNS acid reagent for determination of reducing sugar” Analytic Chemistry 31(3), 426

Data Analysis for Practical Class 2

Questions to Answer About Practical Work 2:

1. The DNS method is widely used for quantifying reducing sugars. What other sugars, besides glucose, can be quantified using this method? Which sugars cannot be quantified?

2. Why was glucose selected for the construction of the calibration curve? Could another sugar (like fructose) have been used to construct this curve?

3. Using only the results obtained in this practical class, can you conclude anything about the Pasteur effect?

4. Explain the differences found in glucose levels quantified by the HPLC and DNS methods. What are the advantages of each?

In this work, glucose, ethanol, and intermediate metabolites such as acetic acid, succinic acid, and glycerol will be evaluated over time under different experimental conditions. These compounds can be quantified by different methods: chemical, physical, or enzymatic. In these practical classes, we will use two different methods to quantify glucose:

• Separation by HPLC (High Performance Liquid Chromatography), where different compounds will be separated according to retention time in a column with a specific resin for sugars/acids. With this method, we will quantify not only glucose but also all other mentioned components simultaneously (Practical Class 1).
• Determination of reducing sugars using the dinitrosalicylic acid (DNS) method (Practical Class 2).

1. Determine the levels of glucose, ethanol, acetic acid, succinic acid, and glycerol in Saccharomyces cerevisiae and Torulaspora delbrueckii cells grown under different experimental conditions.

Data Analysis for Practical Class 1 (To be conducted in Practical Class 2)

After obtaining the data from the HPLC, this class will introduce the separation method by HPLC and analyze the results obtained from the previous class. After the explanation of the HPLC method and result analysis, each student must perform all necessary calculations, construct the requested graphs, and submit them for evaluation.

Procedure:

1. Obtain the data related to the quantification of standard solutions of glucose, ethanol, acetic acid, succinic acid, and glycerol that were previously prepared and run on the HPLC (provided by the teacher).

2. Using the previous data, construct the calibration curve using Excel for each of the standard solutions.

3. Using the calibration curves, calculate the concentration of glucose, ethanol, acetic acid, succinic acid, and glycerol, in mg/mL, obtained with Saccharomyces cerevisiae and Torulaspora delbrueckii, and construct graphs of the concentrations as a function of incubation time.

Questions to Answer in the Notebook:

1. Explain the reason for the different kinetics obtained for each quantified compound, comparing the values obtained in the presence and absence of oxygen. Explain the effect of oxygen on the kinetics of the different compounds (consider the Pasteur effect).

2. Compare the values obtained with Saccharomyces cerevisiae and Torulaspora delbrueckii cells and conclude regarding the Crabtree effect associated with each species. Besides this effect, is there any other difference in metabolism between these two yeasts?

3. Briefly explain the purpose of metabolite quantification by HPLC and why it was useful for this practical work.

4. Why is it necessary to construct calibration curves for the quantification of compounds by HPLC?

5. What other hypotheses could be tested (and how) in the present practical class to understand the various stages of glycolysis in these yeasts?