Carbohydrates Jennifer Driskell Lab Partners: Laura Dewalt
Date Due: April 5, 2012 Date Submitted: April 5, 2012 Section 004 TA: Saikat Choudhury
Introduction There are two approaches to identifying an unknown component, qualitative and quantitative identifications. The qualitative approach identifies the components but does not specify the amount of each component and the quantitative approach determines both the nature and the amount of each component in the mixture.1 Both qualitative and quantitative approaches will be used in this experiment dealing with carbohydrates. In this experiment, glycogen will be broken down (hydrolyzed) to its glucose monomers. The first part uses a qualitative analysis, determining the products of acid hydrolysis of glycogen from the products of the enzymatic hydrolysis of glycogen. Procedure 1 reacts glycogen with HCL and heat causing dissociation of the glycosidic bonds whereas, Procedure 2 reacts glycogen with the enzyme salivary amylase hydrolyzing the glycosidic bond in a more specific manner. Thin layer chromatography will then be used to analyze all reaction products.1
Materials and Methods Procedure 1 In a large test tube, 1.0ml of 4.0 M HCL is added to 1.0ml of glycogen (100 mg/ml) and then a marble is placed on top of the tube prior to it being placed in a boiling water bath for 30 minutes. After 30 minutes, 1.0ml of the solution in the test tube (acid hydrolysate) is added to 5.0ml of 1.0M K 2HPO4. Using a microcapillary pipet, 5µl of this acid hydrolysate is spotted onto a labeled TLC plate. Procedure 2 My lab partner collected 0.5ml of saliva in a beaker from which a 1/10 dilution was made using distilled water. In a test tube, 1.5ml of distilled water, 1.0ml glycogen (100mg/ml) and 0.5ml of diluted saliva were mixed. After 10 seconds, 1ml of this solution was removed from the test tube and placed into a pre-chilled test tube. Using a microcapillary pipet, 10µl of this 10 second enzyme hydrolysate
were spotted onto our TLC plate. After 30 minutes, the remaining solution was placed on ice and then a 10µl amount was spotted on the TLC plate with a microcapillary pipet. Procedure 3 (performed by TA) The teaching assistant labeled the TLC plate as shown below with faint pencil marks. He then applied 5µl of carbohydrate standards to the plate as shown below. The standards were glucose (G), maltose (M) and maltotriose (T). When the plate was completed spotted the teaching assistant placed the plate in a TLC developing tank containing chloroform: glacial acetic acid: water (30:35:5) and then removed the plates 3-4 hours later. The following week, the teaching assistant sprayed chromatogram evenly with aniline diphenylamine. The plates were heated to 100°C for 10-15 minutes. The plate was then copied onto our data sheet. Using the TLC plate, measurements from the origin to the edge of each spot were taken and then used to calculate the retardation factors for each spot. This data can be seen in Table 1 and a sample calculation for Rf value can be seen in Equation 1. Figure 1 TLC Plate Set-up
Results Table 1 Data from TLC Plate This data was collected from the TLC plate. The distance from the origin to the edge of the spots was measured for the three standards and for all of the spots present for the acid hydrolysis and the two enzyme hydrolysis. The solvent front was measured at 17.7cm.
Standards Distance (cm)
Rf
Enzyme Hydrolysis Enzyme Hydrolysis (10 sec) (30 min) Distance Distance (cm) Rf (cm) Rf Spot Spot 0.282 1 0.7 0.039 1 1.0 0.056 Spot Spot 2 1.6 0.090 2 3.5 0.197 Spot 3 5.2 0.293
Acid Hydrolysis Distance (cm) Rf
Glucose
5.5
0.310 Spot 1
Maltose
2.5
0.141
Maltotriose
1.2
0.067
5.0
Equation 1 Calculating Rf Values
Sample calculation for Glucose Standard:
Discussion Maltotriose is closest to the origin with an R f value of 0.067 followed by Maltose with an Rf value of 0.141 and lastly glucose being the farthest away from the origin with an R f value of 0.310. As for the acid hydrolysis, one spot was visible on the TLC plate and it most closely relates to the glucose standard with and Rf value of 0.282. The 10 second enzyme hydrolysis had two spots visible on the TLC plate, although the Rf values are lower than the standards they most closely relate to the Maltose and Maltotriose standards. The 30 minute enzyme hydrolysis had 3 spots and they very closely resembled all three of the standards. Although our spots matched pretty closely to the standards, it is possible that
this procedure could produce spots that do not resemble any of the standards. This can occur due to the nature of how the bonds are cleaved leaving carbohydrates that are larger/smaller than the standards.1 The susceptibility of the glycosidic bond to acid and salivary amylase varied. The bond was more susceptible to the acid than it was to the enzyme salivary amylase as can be seen with the distribution of spots from the TLC plate. For the acid hydrolysis only one spot was visible on the TLC plate which demonstrates that all of the glycogen was broken down to glucose that none of it was partially broken down to maltose or maltotriose. For the enzyme hydrolysis, the 10 second and 30 minute times varied on how well the glycosidic bond was broken down. With only 10 seconds, the enzyme was able to break down the bonds to form maltose and maltotriose but the 30 minute displayed all three components of the breakdown.1 A three hour time point for salivary amylase would not look like the 30 minute time point because at 30 minutes the hydrolysis was not complete. By looking at the Enzyme Hydrolysis (the 30 minute reaction) there were spots on the TLC plate that correlated with the glucose standard but there were also spots that migrated less. The spots that migrated less would represent larger carbohydrates and had similar Rf values as the maltose and maltotriose standards. After 3 hours one would expect the hydrolysis to be complete or just about complete by displaying a prominent glucose spot on the TLC plate and the other spots would be less prominent or absent altogether.1 If a large enough TLC plate that would allow the solvent front to migrate twice as far as it did in this experiment, I would expect the migration distance of the TLC spots from the origin to increase proportional to the solvent front. Since the solvent front is what carries the samples up the TLC plate it would only make sense that if the solvent front went twice as far that the spots would travel twice as far. 1
Salivary amylase functions to break down polysaccharides, particularly starch, into disaccharides, mainly maltose, by breaking 1, 4 glycosidic bonds between the various molecules that make up the polysaccharide. A substrate is the basis or substance on which an enzyme works. In the case of salivary amylase, the substrate is starch, which it reduces to short polysaccharide chains and to maltose and maltotriose, the products. 2 Extra Credit: The carbohydrate that accounts for an extremely large percentage of the biomass is cellulose. Cellulose is a compound that humans cannot utilize because we do not have the particular enzyme cellulase to break down it down into its individual components. There are many animals that can utilize cellulose (which is found in the cell wall of many plants) the primary animals include but are not limited to ruminants like cows and sheep along with many bacteria and fungi. The animals do not actually posses the enzyme needed to break down cellulose but in their stomach they contain many bacteria species that are capable of breaking down cellulose therefore the bacteria and the animal are able to live symbiotically in which the animal provides cellulose and then the bacteria gives the animal unused products from the breakdown.3
References 1
Padala, Dr. Reddy. "Experiment 5- Carbohydrates." Biochemistry & Molecular Biology 212. Spring 2012 Ed. New England Biolabs Catalog, 2005/2006. 37-44. Print.
2
Salivary Amylase. Ed. Tilottama Chatterjee. Buzzle.com, 28 Sept. 2011. Web. 4 Apr. 2012. .
3
Cellulose. Wikipedia, 28 Mar. 2012. Web. 4 Apr. 2012. .
