b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 7 4 8 e4 7 5 0

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Short communication

Differential behaviour of the dinitrosalicylic acid (DNS) reagent

towards mono- and di-saccharide sugars
Abdul Aala Najmus Saqib*, Philip John Whitney
Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, UK

article info

abstract

Article history:

3,5-Dinitrosalicylic acid (DNS) reagent is widely used in the estimation of reducing sugars.

Received 29 June 2009

The reagent shows a differential behaviour towards mono- and di-saccharides. This

Received in revised form

phenomenon has been misinterpreted in the literature. Contrary to the facts, it has been

4 September 2011

reported that the DNS test is less sensitive for the estimation of cellobiose than it is for the

Accepted 12 September 2011

estimation of glucose. This communication clarifies the concept. In addition, the study also

Available online 29 September 2011

compares the reaction of different mono- and di-saccharides and discusses the difference
in their reactivity.

Keywords:

2011 Elsevier Ltd. All rights reserved.

DNS
Reducing sugars
Lignocellulose
Mono-saccharides
Di-saccharides

1.

Introduction

Utilisation of biomass in future for the production of biofuels

is an area of intensive research [1e4]. Degradation of the
lignocellulosic material to simple utilisable products is the key
step in this process. The product of interest in the biodegradation of cellulose is reducing sugars which can be used in
further fermentation steps for the biofuel production [4,5].
Production of reducing sugars during cellulose degradation
can be monitored in a number of ways [6e9]. Use of DNS
reagent [10] for the determination of reducing sugars is not
only a widely practised method [7,11,12], but, it is also an
assay recommended by the International Union of Pure and
Applied Chemistry (IUPAC); It is also an integral part of the

filter paper assay which is another recommended assay by the

IUPAC for the measurement of cellulase activities [8].
Sugar molecules act as reducing agents as long as they
contain an aldehyde group and exist in an open chain structure. Monomeric sugars exist in aqueous solution in equilibrium between their open chain and ring structures but only
the open structures are responsible for their reducing activity.
During dimer formation the aldehyde group of one of the
sugars is buried in the glycosyl bond making it incapable of
acting as a reducing agent anymore. On the other hand, one
more reducing end would be produced if a disaccharide, such
as cellobiose, is hydrolysed. The cellobiose contains 2 glucose
rings, but in aqueous solution only 1 ring is opened, hence,
one reducing end is present in intact cellobiose. Each

* Corresponding author. Present address: Food and Biotechnology Research Centre, PCSIR Labs Complex, Ferozpur Road, Lahore 54600,
Pakistan. Tel.: 44 92 3317780266; fax: 44 92 42 9230705.
E-mail address: a.saqib@gmail.com (A.A.N. Saqib).
0961-9534/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biombioe.2011.09.013

4749

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 7 4 8 e4 7 5 0

2.

Materials and methods

All chemicals were purchased from Sigma Aldrich Co., St

Louis, MO (USA), except maltose & lactose (BDH Ltd., Poole,
England); Glucose (Fisher Scientific UK Ltd., Loughborough,
UK).

2.1.

Preparation of DNS reagent

DNS reagent was prepared according to Coughlan & Moloney

[14]. 10 g of dinitrosalicylic acid (DNS) and 300 g of sodium
potassium tartrate (Rochelle salt) was added to 800 mL of 0.5 N
NaOH and was gently heated to dissolve the reagents. The
volume was then made up to 1.0 L with distilled water.

2.2.

Reducing sugar assay

Reducing sugars were assayed in solution as described by

Wood and Bhat [9].
Sugar solutions were prepared in RO (reverse osmosis)
water. To 1 mL of the sugar solution in a test tube 4 ml of the
DNS reagent was added. Tubes were placed in boiling water
bath for 5 min, transferred to ice to rapidly cool down and then
brought to room temperature by placing them in water bath at
25  C. The absorbance was measured at 540 nm, using Pharmacia Biotech Novaspec II spectrophotometer.

2.3.

Statistical analysis

All experiments were performed in triplicate and error bars

for standard deviation from the Mean are shown on the
figures. The error bars may be obscured in some figures
because the standard deviation was too small in those cases.

3.

Results and discussions

As the molecular masses of cellulose or its hydrolysis products are variable, the concentration of reducing sugars is
usually measured in terms of g/L glucose equivalent to estimate the extent of saccharification. This can be misleading in

1.2

y = 0.1106x + 0.0067

Absorbance at 540 nm

Glucose

1
y = 0.09x - 0.0086

0.8
Cellobiose

0.6
0.4
0.2
0
0

10

Sugars (milligram per ml)

Fig. 1 e Reaction of DNS with equal concentrations (w/v) of

glucose and cellobiose.

certain cases. For example, the DNS has been claimed to be

less sensitive towards cellobiose than glucose [15] which, in
fact, is not the case. The falsification, in this particular case,
was the result of making sugar solutions in g/L concentrations. A given weight of cellobiose, a dimer of glucose, would
contain fewer molecules (thus less reducing groups) as
compared to glucose of the same weight. That is why DNS
would appear less sensitive towards cellobiose than towards
glucose when the sugar solutions are made in g/L (or mg/ml)
concentrations. Fig. 1 depicts this observation where sugar
solutions were made in terms of mg/ml.
When equimolar quantities of both of the reducing sugars
were used (Fig. 2) there would have been equal number of
reducing groups present in the solution. Ideally, the graphs for
both of the sugars should coincide. But, we observed a significantly greater slope for cellobiose than glucose ( p < 0.006).
From these results the conclusion can be drawn that either the
cellobiose molecule gives a stronger colour reaction with DNS
[hypothesis 1] or is hydrolysed to some extent under the
influence of DNS reagent [hypothesis 2]. Miller [10] has
pointed out earlier in favour of the second hypothesis that
some of the reducing sugars may hydrolyse during the assay.
In order to test the two hypotheses we used three disaccharides, namely maltose, lactose and cellobiose. DNS consistently gave greater colour with disaccharides than with
monosaccharides (Fig. 3). As the type of monomers and nature

Absorbance at 540 nm

cellobiose molecule, however, upon hydrolysis splits up into

to two glucose monomers and 2 reducing ends are produced.
Therefore, an assay based on the measurement of increase in
reducing sugar equivalents would give a direct estimation of
the number of glycosidic bond cleavages and, thus an estimation of enzyme activity where applicable. The proposed
basis of the DNS reaction is the reduction of 3,5dinitrosalicylic acid (DNS) to 3-amino-5-nitro-salicylic acid
(ANS) and, during this process, the aldehyde group of sugars is
oxidised to the respective carboxylic acid [13].
Correct estimation of cellobiose, a disaccharide and
glucose, a monosaccharide, is the key to determine the
enzyme activities in many cases [4,9]. This short technical
note discusses the differential behaviour of the DNS reagent
towards the estimation of mono- and di-saccharides. In
addition, it also proposes the precautions that must be taken
while making such measurements.

0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Cellobiose

y = 0.3451x - 0.0455

y = 0.2359x - 0.0415

Glucose

0.5

1.5

Sugars (micro moles per ml)

Fig. 2 e The reaction of DNS with equimolar concentrations

of glucose and cellobiose.

Absorbance at 540 nm

4750

b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 4 7 4 8 e4 7 5 0

0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

references

cellobiose
glucose
Maltose
Lactose
fructose
galactose

Disaccharides

Monosaccharides

0.5

1.5

sugars (Micro mole per ml)

Fig. 3 e Variation in the reaction of DNS with

monosaccharides (galactose, glucose and fructose) and
disaccharides (cellobiose, lactose and maltose).

of bonding in these disaccharides was different it is improbable that each disaccharide was partially hydrolysed during
the assay to exactly the same extent. Interpretation of the
results is somewhat complicated by the observation that the
monosaccharides fructose and galactose gave a lower colour
reaction than glucose, p < 0.05 (Fig. 3). As Fructose is a ketose
this difference in behaviour could have been the result of
different functional groups. However the reaction of galactose, an aldose, resembled to that of fructose instead of
glucose (Fig. 3). These findings suggest that colour formation
with DNS and reducing sugars is not exclusively due to
reduction of DNS to ANS (the first hypothesis). This opinion is
supported by the fact that there are small but significant
differences in the absorbance spectra of the reaction products
of different sugars with DNS (not shown) and that these
differed to the spectrum of pure ANS published by Hostettler
et al. [13]. So, Millers [10] concern is still valid that .different
sugars yield different amounts of colour suggest that the
chemistry of the test may actually be appreciably more
complicated.
Therefore, a standard curve for each sugar assayed has to
be constructed separately and results for sugar mixtures
should be treated with caution, and their expression on the
basis of weight per volume or molarity should be chosen
carefully. Elucidation of true mechanism of action of DNS with
reducing sugars and understanding the difference in its
behaviour could be helpful in developing a better detection
system for reducing sugars.

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