Jenalyn D. Osuna

GLYCOGEN ISOLATION, PURITY DETERMINATION OF POLYSACCHARIDES and END GROUP DETERMINATION OF POLYSACCHARIDES I. INTRODUCTION Carbohydrates are one of the major nutritional sources of liv ing organisms. They provide energy for an organism to function properly. Sugars, starches and fibers are the three main types of carbohydrates found in food. Sugars and starches are used by the body as a source of glucose for energy production. In contrast, fibers are not broken down by the body and its major function is to help humans maintain weight and stay healthy (Wax, 2016). Carbohydrates also serve as energy storage, structural support and cell to cell recognition functions. In general, carbohydrates are polyhydroxy aldehydes or ketones, which differ in solubility, properties and functions. They are classified according to the number of monomeric units, number of carbon atoms, type of carbonyl group present (ketose/aldose) and their oxidizability (reducing/nonreducing sugar). Glycogen, amylopectin, and cellulose are some examples of carbohydrates (Nelson & Cox, 2007). Glycogen is defined as a branched polymer of glucose containing minimal amount of phosphate and glucosamine. It is a polysaccharide composed of D-glucose units. Most of them are stored in the liver (6-8%) and skeletal muscles (approximately 1%), which make it readily available for glucose production to the blood stream during period of fasting and to muscle during musc le contraction (Adeva-Andany et. al., 2016). The role of glycogen in maintaining the glucose level in the blood is crucial since glucose is the major metabolic fuels for many organisms. The linkages between glucose residues in glycogen are (α-1→4) and (α-1→6). The structure of glycogen is shown in Figure 1. Figure 1. Chemical structure of glycogen. The first method used in isolating glycogen from a biological source includes heating the sample with 30% aqueous potassium hydroxide solution at 100°C for three hours then precipitating it from the solution using ethanol. The sample was then purified by repeated precipitation with ethanol from the solution. Newer methods were developed because of the degradation of glycogen due to the harsh chemical environment. Scientists developed methods of glycogen isolation in its native or natural form. This result to the use of cold, dilute trichloroacetic acid (TCA) as the extracting solvent. Using the said solvent, it was observed that the mass of the isolated glycogen is ten times more than that of the mass isolated from the previous method. However, it was observed that the molecular weight range of the isolated glycogen is broad, which is due to partial degradation. Figure 2. Structure of dimelthy lsulfoxide (DMSO). Another method developed was the used of dimethylsulfoxide (DMSO) as solvent of extraction. The use of this method gives a result where in the molecular weight of the isolated glycogen molecule is greater compared to the aforementioned methods. However, the yield for this method is relatively lower. This observation indicates the selectivity of DMSO to the easily accessible glycogen with narrow molecular weight range, leaving behind the metabolically active, low molecular weight, residual glycogen. Other extraction solvent used in glycogen isolation includes cold water, aqueous phenol, glycine buffer, and aqueous mercuric chloride. Several experiments show that these newer methods were able to extract glycogen in its native state. It is important to note that the use of hot solvents, acid, and bases should be avoided in extracting glycogen in its native state, since they can cause to the molecule’s degradation. Isolating carbohydrates from tissue samples require purification processes because of the several impurities that come with the isolate. These impurities include proteins, nucleic acids, amino acids, oligosaccharides, and some salt. Trichloroacetcic acid is used in removing proteins from the carbohydrate isolate. It is important to purify the isolate, since it is necessary to gauge the actual amount of carbohydrate present. The purity of the extracted glycogen can be determined by hydrolyzing the sample using an acidic medium to separate it in its monomeric unit (glucose). The amount of glucose present in the solution can be determined quantitatively using Nelson’s assay for reducing sugars. The purity of the isolated glycogen can be calculated using the equation 1. The amount of glucose in the glycogen sample is determined from the am ount of glucose produced upon acid hydrolysis, which is determined using Nelson’s method. This means that the actual amount of glucose is equal to the glucose content of the hydrolyzed sample. The theoretical amount of glucose (µmol) in the glycogen sample is calculated using a conversion factor based from the molar mass of free glucose unit and a glucose residue, which is equal to 180/162. The molar mass of free glucose unit is 180 g/mole, while the mass of the glucose residue is 162 g/mole. In the calculation of the glucose residue mass, the removal of 1 mole of water molecule for every mole of glucose residue in the formation of a glycosidic bond was considered as shown in equation 2. The theoretical amount of µmoles of glucose/mg glycogen can now be calculated using equation 3. Assuming that the isolated glycogen is pure, the amount of glycogen is equal to the amount of glucose residue. Therefore, solving equation 3 gives a theoretical value of 6.17 µmol glucose/mg glycogen. The formula for % purity is now simplified according to equation 4. For the calculation of the actual µmol glucose/mg glycogen, equation 5 will be employed. Starch, like glycogen is composed of polymer of D-glucose units. Amylopectin, a starch isolated from plants has similar structural features to glycogen. Amylopectin also has (α-1→4) and (α1→6) glycosidic bond. Therefore equation used in determining the purity of glycogen can also be used in determining the purity of an amylopectin sample. The structural difference of amylopectin and glycogen can be determined using end -group analysis. Unlike other starch, amylose and cellulose, amylopectin is not linear and has 1,6glycosidic bond branches every 25 glucose units. The linear portion of these branched polysaccharides is made up of (α-1→4) linkage between two glucose residues. Like glycogen, amylopectin has free hydroxyl groups which can react with oxidation reagents. The structure of amylopectin showing the structural features mentioned above is shown below. Figure 3. Structure of amylopectin. Glycogen and amylopectin have reducing and non-reducing ends. The oxidation reaction of the reducing end could be employed in determining the structure of polysaccharides, in general. The degree of branching of a polysaccharide can be determined by three independent methods like methylation technique, periodate oxidation, and enzymatic method (Illingworth, Larner & Cori, 1952). Polysaccharides containing free hydroxyl groups have the ability to react with periodic acid or it’s salt. Vicinal diols (-OH groups) react with periodic acid resulting to two aldehydic groups upon cleavage of the carbon chain. This reaction consumes one molar equivalent of periodate. It is also possible for double cleavage of the carbon chain in both sides of the β-position, which results to the formation of two aldehydic groups and one formic acid. Figure 4. Reaction of a sugar unit with periodate forming two aldehydic groups with cleavage of the carbon chains. Figure 5. Reaction of a sugar unit with periodate consuming two molar equivalent of periodate, which results to the formation of two aldehydic groups and one formic acid. Polysaccharides differ in their linkage patterns, thus they vary significantly in the way they react with periodates. For instance, the non-reducing end of a sugar residue and/or 1,6-linked nonterminal residues contain three adjacent hydroxyl groups, double carbon cleavage will occur resulting to the consumption of two molar equivalents of periodate and the production of one molar equivalent of formic acid. On the other hand, the non-terminal units with 1,2 or 1,4linkage will consume one equivalent of periodate without the formation of formic acid. Sugar units without adjacent hydroxyl groups, such as 1,3-linked residues will not be affected by this reaction. The determination of periodate consumed and the formic acid form ed in the reaction, together with the information on the sugar units surviving the oxidation reaction could be used to deduce the nature of the glycosidic linkage and the structural features of the polysaccharide molecule being studied. The objectives of this exercise is (1) to isolate glycogen from mussel flesh, (2) to determine the yield of the isolation process, (3) to determine the purity of the isolated glycogen and commercial amylopectin, and (4) to determine the structural difference between amylopectin and glycogen using end group analysis. II. METHODOLOGY A. Glycogen Isolation Mussel freshly brought from the market was used as glycogen source in this exercise. The flesh was separated from the shell and was placed in an ice bath. After cooling, about 40 grams of homogenized flesh was weighed in an Erlenmeyer flask. At the same time, 30% potassium hydroxide solution was transferred to a 125-mL Erlenmeyer flask. For every 40 grams of the mussel flesh sample, 7.20 mL of 30% KOH solution was added. The hot potassium hydroxide solution was transferred in the flask containing the flesh homogenate. The mixture was heated in hot water bath for about an hour until all the sample was completely homogenized and digested. Constant swirling was done to hasten the saponification process. The mixture was diluted with 15.0 mL water and transferred to a larger Erlenmeyer flask. About 30.0 mL of 95% ethanol was added next and then the mixture was swirled. The flocculent precipitate formed is principa lly glycogen contaminated with some proteins. The mixture was allowed to stand and was cooled to room temperature. It was placed in an ice bath for 20 minutes, allowing complete separation of glycogen to occur. The speed of the centrifuge was set to half the maximum speed. The precipitate was collected by centrifugation for five minutes. The mass of the centrifuge tubes was ensured within 0.1 g of each other. The supernatant was then discarded. The glycogen collected was dissolved in 6.00 mL cold 10% trichloroacetic acid solution, which was centrifuged to remove contaminants like the brown protein residues. The supernatant was retained, and the glycogen was recovered from it. 15.0 mL of 95% ethanol was added to the supernatant. It was placed in an ice bath for 10 minutes, and the formed precipitates were collected after centrifugation. The white precipitates were dissolved in 4.00 mL water and were reprecipitated by adding 5.00 mL of 95% ethanol. After isolating the glycogen, its weight was obtained. The amount of isolated glycogen was determined and was expressed as grams glycogen per ten grams of the mussel flesh sample. B. Purity Determination of Polysaccharides The first part of the experiment was the preparation of the reagents needed in the exercise. About 20 mM glucose stock solution was prepared. Approximately 0.1800 g glucose was weighed in a 100-mL beaker, which was dissolved using 20 mL distilled water. The solution was quantitatively transferred in a 50-mL volumetric flask, then diluted to mark. For the preparation of 2 mM standard glucose solution, 5.00 mL of the prepared 20 mM glucose stock solution was diluted to 50 mL in a volumetric flask using distilled water. Nelson’s reagent was prepared by mixing 50.0 mL solution A and 2.00 mL of solution B. Solution A of the Nelson’s reagent is composed of one liter solution containing 25.0 g sodium carbonate, 25.0 g sodium potassium tartrate, 20.0 g sodium bicarbonate, and 200.0 g sodium sulfate. Solution B contains one hundred milliliter of solution containing 15.0 g copper (II) sulfate pentahydrate and two drops of concentrated sulfuric acid. For the preparation of the arsenomolybdate reagent, a mixture of 50.0 g ammonium molybdate, 42.0 mL concentrated sulfuric acid, and 6.0 g sodium arsenate were mixed and diluted to one liter of solution. 10 mg/mL glycogen solution was prepared by dissolv ing 50 mg of isolated glycogen using 5.00 mL distilled water in a 50-mL beaker. Lastly, 10 mg/mL amylopectin solution was made by adding 5.00 mL distilled water to 50 mg amylopectin in a test tube covered with marbles. Then the mixture was heated slightly in a boiling water bath until all amylopectin were dissolved. The calibration curve was prepared in this exercise. Eight test tubes were prepared containing varying amounts of 2mM glucose solutions. Test tube 1 served as the blank test tube. Test tubes 2 to 8 have contained 0.050 mL, 0.100 mL, 0.200 mL, 0.400 mL, 0.600 mL, 0.800 mL and 1.000 mL 2mM glucose solution respectively. Different amounts of distilled water were added to each test tubes. Test tube 1 was added with 1.000 mL distilled water, while no distilled water added to test tube 8. Test tubes 2 to 7 was added with 0.950 mL, 0.900 mL, 0.800 mL, 0.600 mL, 0.400 mL, and 0.200 mL distilled water, respectively. Exactly 1.00 mL of the prepared Nelson’s reagent was added to all test tubes. The test tubes were covered with marbles and were heated in a water bath for 20 minutes. After heating, the test tubes were cooled to room temperature. Exactly 1.00 mL of asenomolybdate reagent was added to all test tubes. The contents of each test tube were mixed using the vortex mixer. Afterwards, they were stood for 5 minutes at room temperature. About 7.00 mL of distilled water was added to each test tube, and was mixed using the vortex mixer. It was noted that the standard solutions and samples were assayed simultaneously using the Nelson’s method. The absorbances of the samples in the test tubes were read at 510 nm. T he calibration curve was made by plotting A510 with glucose concentration (µmol/mL). Glycogen and amylopectin undergone acid hydrolysis. Eight test tubes were prepared for the acid hydrolysis of glycogen. Test tube 1 and 2 was added with 0.400 mL and 0.600 mL distilled water, respectively. No distilled water added in test tubes 3, 4, 5, 6, 7, and 8. 0.400 mL of 10 mg/mL glycogen was added to test tubes 2 to 8. Test tube 1 served as the blank and no glycogen added to it. 0.600 mL of 2 N hydrochloric acid was added to test tubes 1 and 3 to 8. 1.2 N sodium hydroxide solution was added next. 1.00 mL of NaOH was added to test tube 1 and 2, while the other test tubes do not contain NaOH at all. Each test tube was covered with a marble. Test tubes 1 and 2 were stood at room temperature, while test tubes 3 to 8 were placed in a boiling water bath. Starting from test tube 3, each tube was removed from the water bath at 5 minutes interval. Test tubes 1 and 2 were not heated. Test tube 3 was heated for 5 minutes, test tube 4 for 10 minutes, 5 for 15 minutes, and so on. For the heated samples, the reaction was terminated by addition of 1.00 mL of 1.2 N NaOH after the specified heating time. All test tubes were added with 8.00 mL distilled water. The contents of the test tubes were mixed using the vortex mixer. The same procedure was used for the acid hydrolysis of amylopectin. The glucose content of the hydrolyzed samples, glycogen and amylopectin was determined. About 0.50 mL aliquot from each of the diluted samples were obtained from the acid hydrolysis of glycogen and amylopectin. Each aliquot was diluted to a final volume of 1.00 mL distilled water. The test tubes were mixed well and each solution were assayed including the blank with Nelson’s method. The protocol from the preparation of calibration curve was applied using the prepared samples in place of the standard glucose solutions. The absorbances were read at 510 nm using test tube 1 as the blank for both acid hydrolysis of glycogen and amylopectin. C. Preparation of Glycogen and Amylopectin Samples for End Group Determination of Polysaccharides Two hundred milligrams of sample was weighed in a 100-mL beaker. About 20.00 mL of 3% NaCl was weighed and added to the sample (glycogen or amylopectin). The mixture was heated gently on a warm water bath. The solution was transferred quantitatively to a 50-mL volumetric flask using approximately 2.00 mL of distilled water. 10.00 mL of 0.350 M sodium periodate was added to the transferred glycogen solution followed by the adjustment of the volume to 50.00 mL using distilled water. A blank sample was prepared in the similar way but was done without the addition of the sample in the mixture (glycogen or amylopectin). The solution was placed in the dark. The prepared solutions were placed inside the refrigerator until use. Five milliliters of aliquots of periodate were transferred to 125-mL Erlenmeyer flask. Three drops of ethylene glycol was added to the solutions, which were placed in dark for 15 minutes. The sample was titrated with standardized 0.01 N NaOH solution, using phenolphthalein as the indicator. Three trials were made. III. RESULTS AND DISCUSSION The first part of this experiment is the isolation of glycogen, a multi-branched polysaccharide composed of glucose units. It is a form of glucose that is readily mobilized. Glycogen is an important energy storage in the human body. This molecule is broken down in the body to release glucose, and increase its amount between meals. Therefore, glycogen serves as a buffer to maintain blood-glucose levels. Maintaining the level of glucose in the blood is important, because glucose is the only source of energy for the brain, except in times of starvation. In addition, the mobilization of glucose from glycogen is a good source of energy for sudden strenuous activity. In this exercise, the source of glycogen is mus sel flesh, because skeletal muscles together with the liver are the two major sources of glycogen in a liv ing organism. The concentration of glycogen in the liver and muscle is approx imately 10% and 2% by weight respectively. However, more glycogen is present in the muscle due to its greater mass (Berg et. al., 2002). This was considered in choosing mussel flesh as glycogen source in the experiment. Approximately forty grams (40 g) of homogenized flesh was obtained and placed in an Erlenmeyer flask and 7.20 mL of 30% potassium hydroxide (KOH) solution was transferred into the flask, and then was heated in a boiling water bath. Potassium hydroxide was added because it is the principal reagent for homogenization of the glycogen isolate. Early methods used by scientist suggest that lesser amounts of glycogen were extracted from animal tissues with water or cold trichloroacetic acid solution compared to the amount obtained when the sample is boiled in an alkaline solution (Roe et. al., 1960). However, they observed that glycogen molecules are destroyed forming low molecular weight fragments, when concentrated potassium hydroxide was used (Barber et. al, n.d.). As a result of the optimization done by early researchers, it was found out that high amounts of glycogen are isolated when 30% potassium hydrox ide solut ion is used (Roe et. al, 1960). Glycosidic bond in glycogen was found out to be resistant to hydrolysis at high temperatures. It is in contrast with the observed reaction of peptide bonds in proteins, ester bonds in lipids and phosphodiester bonds in ribonucleic acids, which hydrolyze at high temperatures and in alkaline pH. In these conditions, glycogen, slightly contaminated with only a very few amounts of other polysaccharides, fragments of denatured DNA and low molecular weight compounds, can be obtained (Glycogen, 2013). Therefore, addition of KOH solution made sure that the glycogen molecules are intact, even though other contaminants are present in the solution. Heating was stopped the moment when there were no clumps of flesh observed inside the flask. The solution underwent constant swirling for the saponification process to accelerate. The mixture was then diluted with 15.0 mL water, and then was transferred to a larger flask. After this, 95% ethanol was added to the reaction mixture, allowing the precipitation of a relatively purified glycogen isolate. This was the reason for the appearance of a flocculent precipitate in the mixture, which is glycogen with some protein contaminants. The mixture was then allowed to stand and cool to room temperature, and then it was placed in an ice bath for 20 minutes to allow the complete precipitation of glycogen to take place. The sample was centrifuged for 5 minutes to collect the precipitate. The supernatant liquid was discarded. The glycogen collected was dissolved in 6.00 mL if cold 10% trichloroacetic acid (TCA) solution. It was followed by the addition of 95% ethanol to dissolve the sample and reprecipitate it from the solution. TCA was used in the experiment because of its ability to remove proteins from the isolated glycogen sample. The removal of protein contaminants in the glycogen isolate is manifested by the disappearance of the brown protein residue after centrifugation. Several factors affect the amount of glycogen isolated in a sample. First is the amount and efficiency of the aqueous KOH solution added. The amount of KOH added is critical since it clumps the glycogen together in the solution. If not enough, not all glycogen present in the solution mixture will be extracted. The amount of ethanol added also affects the yield of the isolation process. Ethanol precipitates the clumped glycogen in the mixture, so the efficiency the precipitation process is directly proportional to the amount of glycogen obtained. Last fact or is the amount and efficiency of TCA solution added. TCA removes protein contaminants from the isolated glycogen sample. The observations in the experiment are presented Table 1. Table 1. Observations on glycogen isolation from mussel flesh. Reagents/Actions Observations Flesh of mussel Soft orangey flesh Homogenized flesh Brown mass of fluid Upon addition of 30% KOH Dark brown mass of fluid After heating for 1 hour Dark brown fluid Upon addition of 95% EtOH Bubbling occurred at the top fluid surface After standing and cooling in ice bath Bubbling was reduced Centrifugation 1  Supernatant Dark brown liquid  Residue Brown mass like mud Upon addition of 10% TCA solution Gray mixture Centrifugation 2  Supernatant Grayish liquid  Residue Dirty gray-white precipitate Upon addition of 95% EtOH to supernatant White residue; clear brown liquid After standing for 10 minutes in ice bath White residues at the bottom Centrifugation 3  Supernatant Light yellow  Residue White solids Upon dissolv ing residue to 4.00 ml dH2O Clear viscous mixture Upon addition of 5.00 ml 95% EtOH Milky white mixture Upon calculation, the amount of glycogen isolated from 10 grams of mussel flesh is 0.0753g. The percent glycogen yield per 10 grams of mussel flesh is 0.753%. The result mentioned was derived from the data presented on Table 2. Table 2. Data for the calculation of percent glycogen yield per 10 grams of mussel flesh sample. Parameters Mass (grams) Homogenized mussel flesh sample 40.0070 Beaker 50.6089 Beaker + glycogen 50.5336 Mass Sample 0.0753 As discussed above, glycogen is important in the body of a liv ing organism. However, a number of inborn errors of glycogen metabolism exist as a result of genetic mutation for the proteins involved in glycogen synthesis, degradation, or regulation. Those disorders, resulting to the abnormal storage of glycogen are known as glycogen storage diseases (GSDa) (Craigen & Darras, 2014). GSDs therefore, are group of inherited diseases resulting to the abnormal functioning, or lack of one of the proteins involved in the conversion of glucose to glycogen or the breakdown of glycogen back to glucose. This disease may lower the normal glycogen levels in the body, if the protein affected is the one that converts glucose to glycogen, or lowering of the glucose level in the blood and building up of glycogen in the muscles and liver if the protein that breaks down glycogen is affected by the disease (Glycogen Storage Disorders, n.d.). There are several types of GSDs but only five of them will be discussed in this paper. Von Gierke’s Disease is the result of the deficiency of the enzyme glucose-6-phosphatase. The said enzyme is present in liver and kidney cell but not in muscle or brain cells. It converts glucose-6-phosphate to glucose. This disease causes the accumulation glycogen in the liver of the von Gierke’s patients since it is not catabolized by the body. High glycogen and glucose-6phosphateconcentration in the cell interior create an osmotic pressure gradient across the cell membrane, which bloats the cell with water. As a result, patients d iagnosed with this disease has enlarged liver, and in serious threat because liver cells begin to rupture (Garrett & Grisham, 1995). Pompe’s disease is a defined as lysosomal-1,4-glucosidase or acid maltase deficiency. This disease result to a buildup of undigested glycogen in lysosomes. This buildup prevents the normal function of the cells. Lysosomes are distributed throughout all types of tissues, a nd all major organs making them so much affected by the said condition. Patients diagnosed usually have swollen heart. Another type of disease is known as McArdle’s diseases, which is the deficiency in glycogen phosphory lase. The deficient enzyme attacks glycogen at its nonreducing end to release glucose-1-phosphate. Glucose-1-phosphate is converted to glucose-6-phosphate n muscle cells, which proceeds to the glycolytic pathway (Garrett & Grisham, 1995). Andersen disease is another example of glycogen storage disease. It is caused by the deficient activity of the glycogen-branching enzyme. It results to the accumulation of abnormal glycogen in the liver, muscle and other tissues. Patients with Andersen’s disease fail to grow and gain weight, and have abnormally large liver and spleen. This condition and lead to liver failure and eventually death (Andersen Disease (GSD IV), n.d.). Type IX Glycogen Storage Disease is a disorder in which the body could not break down glycogen. These glycogen molecules get stored in body organs like liver, muscle, and rarely heart, instead of being used. Patients with type IX GSD could not catabolize glycogen because of the deficiency of the enzyme, Phosphorylase Kinase. This enzyme regulates the amount of glycogen synthesize, so without this enzyme, glycogen gets accumulated in the body. This results to the low production of glucose, which affect the normal function of the body. The next part of this exercise is the determination of polysaccharide purity. In this exercise, the purity of the isolated glycogen sample from mussel flesh and commercial am ylopectin were determined. First, a standard curve is constructed was constructed. Eight test tubes were prepared containing varying amount of the standard glucose sample. Nelson’s assay was employed for the determination of the reducing sugar. The absorbance at 510 nm of the prepared solution was determined and presented in Table 3. Table 3. Absorbance of the standard glucose solution using the Nelson’s Assay. GLUCOSE ABSORBANCE AT 510 nm TEST CONCENTRATION TUBE NO. Trial 1 Trial 2 (µmol/mL- Linear Regression Analysis Slope, ml/µmol- y-intercept- Linearity coefficient- Average slope, ml/µmol- Average y-intercept- Upon getting the absorbance, the standard curve is constructed. The curve is used to determine the concentration of glucose in the sample. The standard curve is shown on Figure 6. Absorbance at 510 nm - y = 0.8541x + 0.053 R² = 0.9678 1.200 Trial 1 0.900 y = 0.8602x + 0.0396 R² = 0.9841 0.600 Trial 2 Linear (Trial 1) Linear (Trial 2) - 0.00 0.40 0.80 1.20 1.60 2.00 Glucose concentration (umol/ml) Figure 6. Standard curve using Nelson’s assay for the determination of t he glucose concentration of the samples. To determine the purity of the polysaccharide sample, it must be broken down into its glucose units for Nelson’s assay to determine its concentration. The samples were hydrolyzed using hydrochloric acid. Eight test tubes were each prepared for glycogen and amylopectin samples. The prepared samples were heated in 5 minutes time interval. After heating, the hydrolysis was terminated using sodium hydroxide solution, and the absorbance of each sample was determined using spectrophotometric method. The actual glucose concentration was determined by multiply ing the interpolated glucose concentration with the dilution factor of 2. The table below shows the absorbance at 510 nm and the actual glucose amounts of glycogen. Table 4. Absorbance at 510 nm and the calculated actual glucose concentrations of glycogen heated at different time interval. Test Heating Interpolated glucose Actual glucose tube time concentration concentration A510 No. (min) (µmol/mL) (µmol/mL- - - - - - - - - The heating time was plotted against the actual glucose concentration, which was taken after 30 minutes of heating where most of glycogen molecules are hydrolyzed to glucose units. Actual glucose conc., umol per ml 0.2 0 0 10 20 30 -0.2 -0.4 -0.6 -0.8 -1 -1.2 Heating time, minutes Figure 7. Actual glucose concentration at 5-minute heating time interval of glycogen. The highest amount of glucose concentration should theoretically peak at 30-minute time interval because at 30 minutes time, it was assumed that all of glycogen molecules present in the solution was hydrolyzed to its glucose units. Therefore, beyond 30 minutes it is assumed that the absorbance of the sample will become constant. The calculated theoretical amount of glycogen is 6.17 µmol/mg. The same procedure was done in the analysis of the commercial amylopectin sample. The dilution factor used for the calculation of the actual glucose concentration is 25. The absorbance was obtained at 510 nm, and the result was presented below. Table 5. Absorbance at 510 nm and the calculated actual glucose concentrations of amylopectin heated at different time interval. Test Heating Interpolated glucose Actual glucose tube time concentration concentration A510 No. (min) (µmol/mL) (µmol/mL- - - - - - - - - - - The highest glucose concentration was obtained from the peak of the actual glucose concentration versus heating time graph. Actual glucose conc, umol per ml 0.1 -0.1 0 10 20 30 -0.3 -0.5 -0.7 Heating time, minutes Figure 8. Actual glucose concentration at 5-minute heating time interval of amylopectin. The last part of this experiment is the experimental determination of the structural differences between amylopectin and glycogen. The method used in this experiment is called end-group analysis, which allows the determination of the identity of the reducing end group and the degree of polymerization of polysaccharide (Biermann& McGinnis, 1988). The structure of the polysaccharides were analyzed using their oxidation with periodate. The numbers of the reducing and non-reducing end of the molecules were estimated by determining the amount of formic acid produced via standard sodium hydroxide titration. The data on the standardization of the sodium hydroxide solution is presented in Table 6. Table 6. Data on the standardization of NaOH solution. Mass of Initial Final reading, Vol. NaOH Trial KHP, g Reading, mL mL used, mL- Average Normality of NaOH:- N Normality of NaOH- In this exercise, periodate (IO 4-) was used to cleave the carbon-carbon bonds containing oxidizable functional groups. Periodate oxidation occurs if vicinal diols or α-hydoxycarbonyl group is present in the molecule. For instance, glucose reacts with periodate forming formic acid and formaldehyde. Oxidative cleavage therefore occurs when a hydroxyl group is right next to the carbonyl group of either an aldehyde or ketone. The amount of periodic acid added is also considered, since the presence of high amounts of this acid will lead to over oxidation to occur, thus formation of unwanted acid. This can lead to positive error upon titration. Covering the solution with a carbon paper also prevented over oxidation. A segment is defined as the portion of the polysaccharide molecule that is located between branch points. The number of segments (SA ) is related to the number of non-reducing ends (NA ) according to the equation below. The quantity 1 in the equation is the number of reducing end in a glycogen molecule. It is neglected because the molecular weight of glycogen is very high, and it contains only 1 reducing end and several non-reducing end. As a result the equation above can be simplified into, For the preparation of the samples to be titrated, 200 mg of glycogen and amylopectin are both placed in separate 100 mL beaker. About 20.0 mL of 3% NaCl was added to each of them, and both were heated gently in warm water bath for the dissolution of the s ample in solution. Then, each of them is transferred to a 50 mL volumetric flask. Ten milliliters of 0.350 M NaIO 4 was added, then the volume was diluted to 50.0 mL using distilled water. A blank sample was also prepared. All of the solutions were titrated with standard 0.01 M NaOH solution. The titration data on the amylopectin, glycogen and blank sample are presented in the tables below. Table 7. Data on the titration of blank, glycogen and amylopectin sample with standard NaOH. Burette Blank Glycogen Amylopectin reading, ml Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Initial- Final- Used- Ave. Vol. Used- Table 8. Parameters needed for the end-group analysis of the polysaccharides. Number of non-reducing Sample Number of segments (SA ) end (NA ) Amylopectin- Glycogen- Table 9. Calculated values for the end-group analysis of amylopectin and glycogen. Parameters Amylopectin Glycogen Total moles of glucose Average glucose units per unit segments Number of branch points Degree of branching Glucose per molecule Number of non-reducing ends per molecule Number of segments per molecule Average number of glucose per segment Consider the structure of amylopectin and glycogen in the analysis of the calculated results above. Figure 9. Chemical structure of glycogen and amylopectin. Glycogen is highly branched compared to amylopectin, which should theoretically be what the calculated result for the degree of branching must suggests. The high degree of branching exhibited by amylopectin is linked to the higher portion of (α-1→6) linkages. Theoretically, amylopectin has 24-30 glucose residue per segment while glycogen has 8-12 glucose residues per segment. The calculated results for the number of glucose residue for both polysaccharides are far from the theoretical value. This is because of the limitations of the procedure used in the experiment. Several measurements are made and the possibility of error committed is high. IV. SAMPLE CALCULATION Part 1 1. Amount of glycogen isolate 2. Percent yield of glycogen isolate per 10 g of sample Part 2 1. Calculation of the glucose concentration (test tube 3) 2. Standard curve construction: Linear regression analysis Linear Regression Analysis Slope, ml/µmol- y-intercept- Linearity- Average slope, ml/µmol- Average y-intercept-. Interpolation of glucose concentration from the standard curve (amylopectin, test tube 3, 5 minutes heating time) 4. Calculation of the actual glucose concentration (amylopectin, test tube 3, 5 minutes heating time) 5. Calculation of the theoretical µmol glucose/mg glycogen Since, at 100% purity total mass of glucose residue = mass of glycogen 6. Actual glucose concentration 7. Purity determination Part 3 1. Standardization of NaOH solution (trial 1) 2. Number of non-reducing ends 3. Number of segments V. REFERENCES 1. Barber, A., Harris, W., & Anderson, N. (n.d.). Isolation of Native Glycogen by Combined Rate-Zonal and Isopycnic Centrifugation. Biology Div ision, Oak Ridge National Laboratory, and technical Div ision, Oak Ridge Gaseous Diffusion Plant. Oak Ridge, Tennessee. 2. Barber, A., Harris, W., & Padilla, G. (1965). Studies of Native Glycogen Isolated From Synchronized Tetrahymena Pyriformis (HSM). Department of Zoology, University of Carolina. Los Angeles, USA. 3. Berg, J., Tymoczko, J., & Stryer, L. (2002). Biochemistry. 5 th ed. W.H. Freeman, New York, USA. 4. Beirmann, C. & McGinnis, G. (1988). Analysis of Carbohydrates by GLC and MS. CRC Press. 5. Biochemistry of Glycogen. (n.d.). 6. Craigen, W. & Darras, B. (2014). Other disorders of glycogen metabolism: GLUT2 deficiency and aldolase A defieciency. 7. Glycogen. (2013). 8. Illingworth, B., Larner, J., & Cori, G. (1952). Structure of Gly cogens and Amylopectins: I. Enzymatic Determination of Chain Length. Department of Biological Chemistry. Washington University School of Medicine 9. Roe, J., Bailey, J., Richard Gray, R., & Robinson, J. (1960). Complete Removal of Glycogen from Tissues by Extraction with Cold Tichloroacetic Acid Solution. Department of Biochemistry, School of Medicine, George Washington University, Washington, D. C. 10. Wax, E. (2016). Carbohydrates. Medical Plus. U.S. National Library of Medicine.