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
Article history: Received 29 June 2009 Received in revised form 4 September 2011 Accepted 12 September 2011 Available online 29 September 2011 Keywords: DNS Reducing sugars Lignocellulose Mono-saccharides Di-saccharides

abstract
3,5-Dinitrosalicylic acid (DNS) reagent is widely used in the estimation of reducing sugars. The reagent shows a differential behaviour towards mono- and di-saccharides. This phenomenon has been misinterpreted in the literature. Contrary to the facts, it has been reported that the DNS test is less sensitive for the estimation of cellobiose than it is for the estimation of glucose. This communication claries the concept. In addition, the study also compares the reaction of different mono- and di-saccharides and discusses the difference in their reactivity. 2011 Elsevier Ltd. All rights reserved.

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

lter 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

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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.

1.2
Glucose

y = 0.1106x + 0.0067

Absorbance at 540 nm

1
y = 0.09x - 0.0086

0.8
Cellobiose

0.6 0.4 0.2 0 0 2 4 6 8 10 Sugars (milligram per ml)

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

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 Scientic 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.

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 falsication, 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 inuence 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
0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.5

All experiments were performed in triplicate and error bars for standard deviation from the Mean are shown on the gures. The error bars may be obscured in some gures because the standard deviation was too small in those cases.

Absorbance at 540 nm

2.3.

Statistical analysis

Cellobiose

y = 0.3451x - 0.0455

y = 0.2359x - 0.0415

Glucose

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 saccharication. This can be misleading in

1.5

Sugars (micro moles per ml)

Fig. 2 e The reaction of DNS with equimolar concentrations of glucose and cellobiose.

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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 0

Absorbance at 540 nm

cellobiose glucose Maltose Lactose fructose galactose

references
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 ndings suggest that colour formation with DNS and reducing sugars is not exclusively due to reduction of DNS to ANS (the rst hypothesis). This opinion is supported by the fact that there are small but signicant 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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