Laboratory Experiment pubs.acs.org/jchemeduc
Carbohydrate Analysis Experiment Involving Mono- and Disaccharides with a Twist of Glycobiology: Two New Tests for Distinguishing Pentoses and Glycosidic Bonds Chelsea Herdman, Lamine Diop, and Michael Dickman* Université de Saint-Boniface, Winnipeg, Manitoba R2H 0H7, Canada S Supporting Information *
ABSTRACT: Carbohydrate analysis is an excellent way to expose second-year biochemistry majors to the subtlety and nuance of sugar chemistry. In one, 3-h practical period, students must identify an unknown mono- or disaccharide from a collection of three hexoses (glucose, galactose, and mannose), two pentoses (ribose and arabinose), and three disaccharides (maltose, cellobiose, and lactose). Two new tests have been developed: an enzyme assay with α-glucosidase followed by the Barfoed test quickly distinguishes maltose and cellobiose and 1,1-diphenylhydrazine is used to identify arabinose. The exercise is presented as a way to sensitize new students of biochemistry to the communicative aspect of sugars.
KEYWORDS: Second-Year Undergraduate, Biochemistry, Laboratory Instruction, Hands-On Learning, Inquiry-Based/Discovery Learning, Carbohydrates, Student-Centered Learning
W
possibilities. They are evaluated based on the entire process of identifying their unknown as well as a series of questions that extend the exercise into the realm of glycobiology.5 To encourage an inquiry-based approach by the students, the basic parameters of the experiment are given in the form of a handout more than one week prior to the exercise. These consist of the names of the eight sugars and short descriptions of the seven tests the students can use. Which specific tests they decide to use and the order they choose to conduct them are for the students to determine. Before the actual experiment, each student is required to submit a plan in the form of a flowchart, and the instructor either recommends it or does not. If a “not recommended” is given, an explanation is included. To achieve success, the students must reflect on what needs to be done to arrive at their goal in the limited time allotted. It was deemed important to include mannose, at least two pentoses and an alpha−beta glycosyl bond distinction into the experiment to address the glycobiology aspect. To that effect, the traditional osazone test with phenylhydrazine6 was modified to take advantage of the unique insolubility of mannose phenylhydrazone7 as a specific test for mannose. A test for arabinose was developed with 1,1-diphenylhydrazine8 to complement the general Bial test for pentoses. Finally, a
ith the increasing importance of glycobiology, there is a need to expose biochemistry students to the marvelous diversity of sugar chemistry. The subtle structural differences between monosaccharides explain, in part, the polyvalent nature of sugars, which goes to the heart of many glycobiological phenomena. Carbohydrate analysis, a wellknown component of many biochemistry laboratory programs, employs a number of chemical reactions that are specific to certain sugars in order to distinguish them. However, most published protocols involve the identification of mono- or disaccharides,1−4 and only one procedure involves making distinctions between mono- and disaccharides using just five sugars in total.4 As part of a program of second-year practical exercises for biochemistry majors, a qualitative carbohydrate analysis experiment has been developed using eight sugars (three hexoses, glucose, galactose, and mannose; two pentoses, ribose and arabinose; and three disaccharides, maltose, lactose, and cellobiose) that highlights the many differences between hexoses and pentoses as well as the alpha−beta distinction in typical glycosidic bonds. This exercise is always conducted during the winter term of the second year in order for the students to acquire as much organic chemistry and biochemistry instruction as possible. For the experiment, the students are given a single, three-hour laboratory period during which they analyze an unknown sugar and identify it from eight © 2012 American Chemical Society and Division of Chemical Education, Inc.
Published: October 9, 2012 115
dx.doi.org/10.1021/ed300149s | J. Chem. Educ. 2013, 90, 115−117
Journal of Chemical Education
Laboratory Experiment
Figure 1. A possible order of testing for an unknown among eight mono- and disaccharides.
■
RESULTS AND DISCUSSION For the first year of this practical exercise, the success rate for the correct identification of the unknown was around 50%. It was obvious that the students had not considered the importance of planning and of employing an efficient use of time. Obliging them to submit a flowchart plan prior to the experiment increased the success rate to over 80%. Misinterpretation of experimental data while writing their report is the principal reason for students’ failure to correctly identify their unknown. The Barfoed, Bial, and mucic acid tests are well-known and were used with only minor modifications from standard protocols.6 A fourth standard protocol using phenylhydrazine is often employed for the conversion of sugars to their respective osazones followed by examination of the crystals.6 It was found that if 1.5 equiv of phenylhydrazine was added to the sugars in this experiment, only mannose phenylhydrazone precipitated out of solution. Thus, it is an effective test for mannose. For students having little opportunity to practice the technique, TLC is a rather difficult test to perform well, but it is useful for confirming a preliminary conclusion. The two new tests, 1,1-diphenylhydrazine and α-glucosidase−Barfoed, were developed to include arabinose and cellobiose in the exercise. All the sugars will react with 1,1diphenylhydrazine, but only arabinose will form an insoluble hydrazone that precipitates. Thus, ribose and arabinose can be distinguished from each other after a positive Bial test. As for inclusion of cellobiose, White and Robyt published an interesting method to identify nine different di- and trisaccharides with α-glucosidase, β-glucosidase, and invertase in conjunction with TLC,2 but their method is time-consuming. It was thought that because the Barfoed test could be used to distinguish between mono- and disaccharides, this same test could be used to evaluate the effect of a glycosidase on a particular disaccharide. This new method, which could easily be modified to include more disaccharides and enzymes, involves
relatively quick and easy protocol was elaborated to distinguish cellobiose and maltose using the specificity of alpha glucosidase.
■
EXPERIMENTAL OUTLINE
■
HAZARDS
The sugars for this experiment consist of three hexoses (glucose, galactose and mannose), two pentoses (ribose and arabinose), and three disaccharides (maltose, lactose and cellobiose). The seven tests for the experiment consist of five which are well-known (Barfoed, Bial, mucic acid, phenylhydrazine, and TLC) and two new tests (α-glucosidase with Barfoed and 1,1-diphenylhydrazine). Figure 1 gives a typical flowchart for the identification of an unknown sugar, using these tests. The Barfoed test allows students to identify whether their unknown is a mono- or disaccharide and is definitely the first choice of most students. If a disaccharide is indicated, then the mucic acid and enzyme tests should be used. If the initial Barfoed test is positive for a monosaccharide, then a few options are available, but the Bial test is definitely the best one. A positive Bial result for a pentose leaves only two choices, and they can be distinguished using the 1,1-diphenylhydrazine test. Finally, a negative Bial result narrows the field to three hexoses that can be identified using the mucic acid and phenylhydrazine tests. The students are encouraged to use TLC to confirm their sugar. This is done by doing a TLC plate with the unknown sugar, the presumed sugar, and one or two other sugars similar to the presumed one.
The Bial reagent contains concentrated HCl and is corrosive. Concentrated nitric acid, used in the mucic acid test, is corrosive, and the test should be done in a fume hood, because it produces toxic nitrogen oxide gases. Phenylhydrazine and 1,1-diphenylhydrazine are toxic. 116
dx.doi.org/10.1021/ed300149s | J. Chem. Educ. 2013, 90, 115−117
Journal of Chemical Education
Laboratory Experiment
Notes
mixing the unknown with alpha-glucosidase for an hour followed by direct addition of Barfoed reagent. If the enzyme has hydrolyzed the sugar, then the resultant monosaccharides give a red precipitate in a few minutes. Maltose is a substrate for the enzyme and so gives a positive result. Neither cellobiose nor lactose shows any evidence of reduction even after 60 min. The development of these tests permitted the inclusion of the three sugars, mannose, arabinose and cellobiose, thus greatly increasing the chances to discuss and explore the world of carbohydrates. The first sugar, mannose, is an important monosaccharide in all asparagine-linked glycans (N-glycans) found in animals and a whole set of very rare genetic diseases associated with N-glycan formation is linked to the metabolism of mannose.9 Glucose is not often found in finished N-glycans, but it is used as an indicator for the protein folding of Nglycoproteins.10 Many glucose and galactose derivatives such as N-acetylglucosamine, N-acetylgalactosamine, and glucuronic acid are found in the extracellular matrix (ECM). The various roles that glucose, galactose, and mannose play in the structure and function of complex carbohydrates is highlighted by the chemical differences used to distinguish them. The addition of the second sugar, arabinose, allows the comparison of the two pentoses and the difference of configuration at carbon-2 between ribose and arabinose. If one so desires, many students enjoy the mental experiment of substituting ribose for arabinose in RNA and deciding how stable it would be. (This question is particularly useful if the catalytic cycle of Ribonuclease A has been studied.) The inclusion of the third sugar, cellobiose, and the α-glucosidase test are opportunities to explore the world of enzymes and the significance of an alpha or beta glycosidic bond between two molecules of glucose. The addition of cellobiose with maltose and lactose opens the door to discussion about digestion and the bacteria that live in our gut. Students usually find such talks interesting and meaningful, because they are able to apply them to their immediate lives. Thus, the integration of this exercise in the second year of a biochemistry major program can serve as a platform for discussing a multitude of topics in carbohydrate metabolism and glycobiology.
The authors declare no competing financial interest.
■
REFERENCES
(1) Senkbeil, E. G. J. Chem. Educ. 1999, 76, 80−81. (2) White, B. J.; Robyt, J. F. J. Chem. Educ. 1988, 65, 164−166. (3) Almy, J. J. Chem. Educ. 2004, 81, 708−710. (4) Malherbe, J. S.; Meyer, C. J. J. Chem. Educ. 1997, 74, 1304−1305. (5) Two decent textbooks on the subject. (a) Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P., Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of Glycobiology, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2009. (b) Taylor, M. E. Drickamer, K. Introduction to Glycobiology, 2nd ed.; Oxford University Press: London, 2006. (6) Plummer, D. T. An Introduction to Practical Biochemistry; McGraw-Hill: London, 1978; pp 106−110. (7) White, L. M.; Secor, G. E. Anal. Chem. 1956, 28, 1052−1053. (8) Secor, G. E.; White, L. M. Anal. Chem. 1955, 27, 1998−1999. (9) Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P., Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of Glycobiology, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2009.; pp 586−592. (10) Taylor, M. E.; Drickamer, K. Introduction to Glycobiology, 2nd ed.; Oxford University Press: London, 2006; pp 156−158.
■
SUMMARY This experiment helps to expose students, relatively new to the field of biochemistry, to the myriad possibilities of sugar chemistry. They learn that most mono- and disaccharides look very similar but do not react in the same way. As part of their report, the students are asked to reread the introduction to carbohydrates in their textbook as well as a short chapter in Essentials of Glycobiology9 and answer a series of questions that emphasize the glycobiological connections to the practical exercise.
■
ASSOCIATED CONTENT
S Supporting Information *
The initial student handout; questions for the students; instructor notes. This material is available via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 117
dx.doi.org/10.1021/ed300149s | J. Chem. Educ. 2013, 90, 115−117
