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 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.

Pasted image 20241120073157-1.png

Reducing sugars

All monosaccharides (Figure 1) and some polysaccharides are reducing sugars, for example:

• ✅ Glucose
• ✅ Fructose
• ✅ Galactose
• ✅ Maltose
• ✅ Lactose
• ❌ Sucrose

A reducing sugar has a free aldehyde or ketone group:

Pasted image 20241120062110-1.png Figure 1

Most sugars in cyclic form containing a hemiacetal (Figure 2) and are reducing since they are in equilibrium with a free aldehyde group.

Pasted image 20241120071712-1.png Figure 2

Most monosaccharides are found in both linear and cyclic form (Figure 3).

Pasted image 20241120063726-1.png Figure 3

Some sugars have only acetals and are therefore not reducing (Figure 4).

Pasted image 20241120064559-1.png Figure 4

Figure 5 - A nice YouTube video on reducing sugars (5 min)

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:

 Figure 6

Experimental Protocol

1. Each group should prepare 4 glass 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 (min)	Volume of Sample	Volume of DNS
1	S. cerevisiae	aerobic	0, 5, 10, 15	0.5 mL	0.5 mL
2	S. cerevisiae	anaerobic	"	"	"
3	T. delbrueckii	aerobic	"	"	"
4	T. delbrueckii	anaerobic	"	"	"

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. (1959). Use of DNS acid reagent for determination of reducing sugar. Analytical Chemistry. Vol. 31 (3). pp. 426-428.

Optional study questions

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?