EXPERIMENT NO.

4A GLUCOSE ASSAY BY DINITROSALICYLIC COLORIMETRIC METHOD

Prepared by Nam Sun Wang Department of Chemical & Biomolecular Engineering University of Maryland College Park, MD 20742-2111 ENCH485

Table of Contents
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Method List of Reagents and Instruments Procedures Notes Questions References Comments

Method
This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5nitrosalicylic acid under alkaline conditions:
aldehyde group oxidation ----------> carboxyl group

reduction 3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not necessary for the color reaction, is added in the reagent to absorb the dissolved oxygen.

The above reaction scheme shows that one mole of sugar will react with one mole of 3,5dinitrosalicylic acid. However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is more complicated than that previously described. The type of side reaction depends on the exact nature of the reducing sugars. Different reducing sugars generally yield different color intensities; thus, it is necessary to calibrate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar, other side reactions such as the decomposition of sugar also competes for the availability of 3,5-dinitrosalicylic acid. As a consequence, carboxymethyl cellulose can affect the calibration curve by enhancing the intensity of the developed color. Although this is a convenient and relatively inexpensive method, due to the relatively low specificity, one must run blanks diligently if the colorimetric results are to be interpreted correctly and accurately. One can determine the background absorption on the original cellulose substrate solution by adding cellulase, immediately stopping the reaction, and measuring the absorbance, i.e. following exactly the same procedures for the actual samples. When the effects of extraneous compounds are not known, one can effectively include a so-called internal standard by first fully developing the color for the unknown sample; then, a known amount of sugar is added to this sample. The increase in the absorbance upon the second color development is equivalent to the incremental amount of sugar added.

List of Reagents and Instruments

A. Equipment
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Test tubes Pipets Spectrophotometer

B. Reagents
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Dinitrosalicylic Acid Reagent Solution, 1% o Dinitrosalicylic acid: 10 g o Phenol: 2 g (optional, see Note 1) o Sodium sulfite: 0.5 g o Sodium hydroxide: 10 g o Add water to: 1 liter Potassium sodium tartrate solution, 40%

Procedures
1. Add 3 ml of DNS reagent to 3 ml of glucose sample in a lightly capped test tube. (To avoid the loss of liquid due to evaporation, cover the test tube with a piece of paraffin film if a plain test tube is used.) 2. Heat the mixture at 90 C for 5-15 minutes to develop the red-brown color.

3. Add 1 ml of a 40% potassium sodium tartrate (Rochelle salt) solution to stabilize the color. 4. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer at 575 nm.
5. 6. 3ml reagent 575nm --->----+ 1ml O.D. Rochelle soln --->----++-------> at | || | +-++-+ | | | | | | | | |____|

7. 8. 9. 10. 11. 12. 13. 14.

| | | +-+--+ | | heat | | --------> | | | | |____| 3ml sample soln

Notes
1. Phenol, up to 2g/l, intensifies the color density. It changes the slope of the calibration curve of absorbance versus glucose concentration but does not affect the linearity. The above procedure yields an absorbance of 1 for 1 g/l of glucose in the original sample in the absence of phenol in the reagent, as opposed to an absorbance of 2.5 for 1 g/l of glucose in 2 g/l of phenol. This property can be exploited to achieve the maximum sensitivity for dilute samples.

Questions
1. How much time was needed for the complete color development? Justify your answer with a plot of color intensity as a function of time. 2. Obtain an absorption spectrum over wavelengths in the visible range (i,e. 400-700 nm). Justify the use of 575 nm chosen in the Procedure. 3. Find the procedures for at least two other methods commonly employed to measure sugar concentrations. List the advantages and disadvantages of these methods.

References
4. Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426, 1959.

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Glucose Assay Forward comments to: Nam Sun Wang Department of Chemical & Biomolecular Engineering University of Maryland College Park, MD 20742-2111 301-405-1910 (voice) 301-314-9126 (FAX) e-mail: nsw@umd.edu

The Chemical Educator, Vol. 9, No. 4, Published on Web 7/7/2004, 10.1333/s00897040802a, 2004 The Chemical Educator

Glucose Assays Revisited: Experimental Determination of the Glucose Concentration in Honey

Laura DeLong Frost
Department of Chemistry, Georgia Southern University, Statesboro, GA 30460, ldelong@georgiasouthern.edu Received April 6, 2004. Accepted June 22, 2004.

Abstract: Routine glucose assays have been incorporated into the biochemistry laboratory curriculum to emphasize the difference between a nonenzymatic assay and an enzymatic assay. Instead of the typical determination of glucose concentration from a random unknown glucose solution, students are asked to determine the glucose concentration in honey. Using honey as the glucose unknown allows students to readily see the difference in the two assays because the nonenzymatic assay reveals all reducing sugars and the enzymatic assay is specific for glucose. In this work the laboratory procedures are described and typical student results are provided.

Glucose assays are important to the biochemistry laboratory curriculum since the sensing of blood glucose levels is critical in the control of diabetes. The colorimetric detection of reducing sugars in solution reflects some of the earliest carbohydrate chemistry available [1]. A dinitrosalicylic acid (DNS) assay has been available since 1955 and is still useful for the quantitative determination of reducing sugars [2]. More recently several enzymatic assays for glucose [3, 4] have

been developed. Georgia Southern University has incorporated both a nonenzymatic and enzymatic glucose assay into its biochemistry curriculum. Biochemistry students are asked to generate standard curves for both assays, determine the concentration of glucose in a glucose unknown (honey), and compare the results. The nonenzymatic assay indicates the presence of all reducing sugars whereas the enzymatic assay is specific for D-glucose. Students will get very different results for the two assays because in addition to glucose, honey also contains the reducing sugar fructose in a ratio of 1.2:1 with the glucose [5]. By comparing the two experiments the students are led to the conclusion that the enzymatic assay gives a much more accurate concentration of glucose in honey, but has the disadvantage of being more costly both in terms of supplies (enzymes must be prepared fresh, kept in a buffered solution, and used during a single laboratory period), time frame, and diligence on their part. Before the laboratory begins, students are asked to provide some background information on the carbohydrate content of honey. Honey contains 23 to 41% glucose and 31 to 44% fructose with a total reducing sugar content (includes 4 to 8% maltose and galactose) of 61 to 84% [5, 6]. After determining the number of milligrams of reducing sugar or glucose present in the honey sample from the standard curve, students are asked to provide the percent reducing sugar and percent glucose present in the honey. Experimental
One three-hour laboratory period is required for each assay. Typically, the nonenzymatic assay is performed in the first laboratory period (the easier of the two laboratories) followed the next week by the enzymatic assay. Both the nonenzymatic and enzymatic assays can be performed using simple bench-top spectrophotometers such as a Spec-20. Micropipettors are used for preparing the assay samples. All chemicals can be purchased from Sigma. The DNS reagent can be prepared for the class in advance; however, the preparation of the reagent is a worthwhile exercise for the students, especially at the beginning of the semester after students have returned from a break. Enzymatic solutions were prepared in advance by the instructor on the day of the laboratory. This was done to save material and minimize waste. It is recommended that the entire enzymatic assay be conducted on the same day with the same solution to minimize any effects of lost enzyme activity. It is instructive for students to prepare their own 0.10 M sodium phosphate (NaH2PO4) buffer, pH 7, to use for dilutions in the enzymatic assay. A standard glucose stock solution is provided to the students for each assay. The students prepare a set of glucose standards ranging from 0.0 to 1.0 mg mL1 (total sample volume 1 mL). Based on the background assignment on sugars in honey, students are also asked to prepare a solution from the honey that should have a concentration in the range of their standard curve for each assay. Nonenzymatic Assay. After adding the DNS reagent, boiling the samples, and diluting with distilled water, samples containing reducing sugar develop a reddish-brown reduction product. Absorbance is measured at 540 nm. Enzymatic Assay. Figure 1 provides a reaction scheme for the enzymatic reaction [3]. This involves the oxidation of glucose to Hgluconolactone using the FAD-dependent enzyme glucose oxidase (eq 1). The glucose oxidase is regenerated in its oxidized form via molecular oxygen to produce hydrogen peroxide (eq 2). The hydrogen peroxide is then used by a horseradish peroxidase to catalyze the oxidation of 2,2d-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), producing a brilliant blue green color (eq 3). The enzymatic solutions are prepared for the students due to cost restrictions. Samples are incubated for 30 minutes in a 37 rC water bath. The absorbance is measured at 725 nm. Students must perform all dilutions using their sodium phosphate buffer instead of the distilled water used in the previous assay. A standard curve is generated for both assays (absorbance vs. mg reducing sugar or glucose) and the concentration of reducing sugar or glucose is determined for the honey solution.

Results and Discussion A representative set of student standard curves for the nonenzymatic and enzymatic assay are shown in Figure 2.
Table 1. Representative Student Results from the Nonenzymatic and Enzymatic Assays of Glucosea
Student 1 2 3 4 Average
a

Percent reducing sugar (nonenzymatic assay) 85.9 69.8 68.8 81.0 76.4 s 8.4

Percent glucose (enzymatic assay) 42.8 35.5 37.0 32.1 36.8 s 4.5

Percentage values are generated from student-calculated values of mg carbohydrate per mg of honey. Students calculate mg reducing sugar or glucose from their standard curve and then divide by the mg honey in each sample and multiply by 100.

(1)
glucose oxidase-FADH2 + O2 p glucose oxidase-FAD + H2O2 (2)

(3)
Figure 1. Reaction scheme for the enzymatic reaction.

Student results of the percentage of glucose found in the honey solution from both assays are shown in Table 1. Individual student values will vary; however, the averages shown indicate that students can get accurate results. At the worst, students should certainly be able to get a smaller value for % glucose than for % reducing sugar. Notes on Student Experimentation One of the most common sources of student error will occur during the micropipetting of the standard glucose and the honey samples. A review of the use of the micropipette prior to the experiment helps alleviate this problem. It should be noted that the enzymatic assay requires the preparation of a phosphate buffer. Some students will forget to use the phosphate buffer and use distilled water as they did for the nonenzymatic assay. The enzymes will not perform optimally in a distilled water solution, which will affect the results. Different floral honeys (e.g., clover, wildflower) can have differing glucose and fructose values. It is suggested that a single brand and variety of honey be used for the entire class. Students tend to have problems preparing a honey solution. Honey is not a substance that is easy to handle. Deciding how

B
Figure 2. Student data set providing standard curves for the (A) nonenzymatic and (B) enzymatic assay of glucose.

much solvent to add to the solute (honey) instead of how much solute to add to the solvent when preparing solutions was unusual, but a benefit for the students. Preparing the honey solution also allowed the students to work through a number of dilution equations when determining the final concentration of glucose present. Conclusion This laboratory exercise illustrates the differences in two glucose assays, both in their specificity and labor intensity. The students generate simple standard curves and assay a honey sample comparing their results to published information on the carbohydrates in honey. The use of honey as the sample for analysis provides a more relevant application of the assay than merely providing the student with a glucose unknown. The materials needed to perform the laboratory are readily available and the equipment is common to most biochemistry laboratories. Acknowlegment. The author would like to thank her CHEM 5542 classes for their data and enthusiasm and Dr. Paul Cerpovicz. Supporting Materials. Students Laboratory Exercise: Nonenzymatic and Enzymatic Measurements of Glucose. These materials can be downloaded in a Zip file (http://dx.doi.org/10.1333/s00897040802a). References and Notes
1. Shriner, R. L.; Fuson, R. C.; Curtin, D. Y. The Systematic Identification of Organic Compounds, 5th ed.; Wiley: New York, 1964; pp 117118. 2. Bernfeld, P. In Amylases, E and F; Colowick, S. P., Kaplan, N. D., Eds.; Methods Enzymol. 1, 1955; pp 149158. 3. Bergmeyer, H. U.; Bernt, E. In Determination with Glucose Oxidase and Peroxidase; Bergmeyer, H. U., Ed.; Methods of. Enzymatic Analysis, 2nd Ed.; 1974; pp 12051212. 4. Kunst, A.; Draeger, B.; Zeigenhorn, J. In UV-Methods with Hexokinase and Glucose-6-Phosphate Dehydrogenase; Bergmeyer, H. U., Ed.; Methods of. Enzymatic Analysis, 3rd. Ed.; 1984; pp 163172. 5. Carbohydrates and the Sweetness of Honey. http://www.nhb.org/download/factsht/carb.pdf (accessed June 2004). 6. The USDA Nutrient Data Laboratory, Search the USDA National Nutrient Database for Standard Reference (keyword Honey) http://www.nal.usda.gov/fnic/foodcomp/search (accessed June 2004).