PRACTICAL 2 Extraction of glycogen from liver cells Objectives
1. To extract glycogen from liver cells. 2. To estimate the quantity of the glycogen by hydrolysis of glycogen into glucose and subsequently determination of the glucose content calorimetrically. 3. To estimate the purity of the extracted glycogen. Materials
Test tubes, centrifuge tubes, centrifuge, oven, pipette, potassium hydroxide, saturated sodium sulphate, ethanol, phenol, sulphuric acid, glucose powder, commercially available glycogen and distilled water. Procedures
As stated in the Biochemistry practical manual. Results
Part 1: Weight of falcon tube: 12.97 g Weight of falcon tube with dried glycogen: 13.50 g Weight of glycogen after extraction: 0.53 g/ 100 g of liver tissues Part 2: Table 1 shows the stock glucose solution and distilled water needed to prepare standard glucose concentration Tube* Standard 0.1 mg/ml Extracted 0.1 mg/ml Distilled water glucose glycogen glycogen (ml) stock glucose (ml) concentration solution (ml) solution (ml) (µg/ml) 1 2 2 2 3 0 2 4 10 0.2 1.8 5 20 0.4 1.6 6 40 0.8 1.2 7 80 1.6 0.4
Table 2 shows the absorbance at wavelength 490 nm for each of the tube Tube* Absorbance at wavelength 490 nm (absorbance unit, A) 1 0.726 2 0.301 *Tube 3 0.000 1: Commercial glycogen solution 4 0.221 2: Extracted glycogen from liver tissues 3: Blank tube 5 0.298 4: Standard glucose concentration 10 µg/ml 5: Standard glucose concentration 20 µg/ml 6 0.350 6: Standard glucose concentration 40 µg/ml 7 0.454 7: Standard glucose concentration 80 µg/ml
Calculation
Given concentration of glucose stock solution 0.1mg/ml, therefore it is 100 μg/ml Tube 4 M1V1 = M2V2 100(V1) = (10)(2) V1= 20/100 V1= 0.20 Therefore, volume of water = 2.00-0.20 = 1.8 ml Tube 5 M1V1 = M2V2 100(V1) = (20)(2) V1= 40/100 V1= 0.40 Therefore, volume of water = 2.00-0.40 = 1.60 ml Tube 6 M1V1 = M2V2 100(V1) = (40)(2) V1= 80/100 V1= 0.80 Therefore, volume of water = 2.00-0.80 = 1.20 ml
Tube 7 M1V1 = M2V2 100(V1) = (80)(2) V1= 160/100 V1= 1.60 Therefore, volume of water = 2.00-1.60 = 0.40 ml ▲These calculations are recorded in Table 1 in th e Results section. Graph
Graph 1 shows standard curve of absorbance versus glucose concentration based on Table 2. (Please refer to the graph attached with this report.)
Tube 1 indicates that the absorbance is 0.726 A and the standard glucose concentration is 106 μg/ml. Tube 2 indicates that the absorbance is 0.301 A and the standard glucose concentration is 44 μg/ml.
Calculation
Weight of glycogen after extraction = 13.50 – 12.97 = 0.53 g/ 100 g of liver tissues Calculate the purity of the extracted glycogen solution: 44 x 10-6g = 44 x 10 -6 / 10-3 = 44 x 10 -3 g = 0.044 mg To calculate the purity in 50 mL of solution, 0.044/2 x 50 = 1.100 mg The percentage of the purity of 50 mL of solution, Convert 0.53 g to milligram 0.53/ 10 -3 = 530 mg 1.100/530 x 100 % = 0.21%
Discussion
Glycogen is stored mainly in liver and muscles. Glycogen accumulated in liver serves as a reservoir that releases glucose into the blood when the blood glucose level falls below the normal one. In this experiment, livers from animals were used to estimate the glycogen content. The first part of this experiment is to extract glycogen from the livers. The liver was digested using hot, concentrated potassium hydroxide (KOH) solution. The reason for using KOH solution is that glycosidic bonds in glycogen are resistant to hydrolytic activity of OH- at raised temperature. On the contrary, peptide bonds in proteins, ester bonds in lipids and phosphodiester bonds in ribonucleic acids undergo hydrolysis at high temperature and in alkaline pH such as KOH solution. Therefore, the glycogen solution can be obtained from this process. On the other hand, the addition of ethanol results in glycogen precipitation and from here, relatively purified glycogen can be obtained. The second part of this experiment is to estimate the glucose content in the extracted glycogen from Part 1. Four standard glucose solutions of 10, 20, 40, 80 µg were prepared in separate tubes. 1 ml of 5% phenol and 5 ml of concentrated sulfuric acid were added into each tube and after cooling the absorbance at wavelength 490 nm was measured. Glucose has reducing properties and when mixed with phenol and concentrated sulfuric acid, it reduced them while itself is oxidized to gluconic acid. After reduction, the mixture changed to orange, with a maximum absorbance at 490 nm. The colour change is directly proportional to the amount of glucose in the sample. After that, a graph of standard curve of absorbance versus glucose concentration is plotted and the quantity of glucose for both the glycogen samples (i.e. commercial glycogen and extracted glycogen.) is determined based on that graph. Next, the purity of the extracted glycogen solution is calculated. Based on the result obtained from the experiment, only 0.21% of pure glycogen was extracted from the liver tissue. This very small amount of purity could be mainly due to the inefficient drying process. This is because the drying process was done using hair dryer and therefore resulting in incomplete removal of all the liquid in the falcon tube. This affects greatly the weight of the extracted glycogen because the reading will be increased due to the remaining liquid content that is not removed. In order to improve the result of this experiment, the glycogen solution must be dried using different method that is to place the falcon tube into the oven and heat the content with high temperature to remove all the unwanted liquid.
While doing this experiment, there are a few precautions that need full attention. For example, while putting the falcon tube in the water bath, take the tube out of the water bath as soon as it starts to boil. This is to avoid the boiling water from entering the tube. Also, mix carefully every time phenol is added by tilting the solution at the wall of the test tubes because the reaction is very vigorous. To do centrifuging, the test tube must be in balanced condition to avoid any accident while the machine is operating. The supernatant must be discarded carefully as well so that the glycogen remains in the tube. Last but not least, gloves and goggle must be worn all the time as there are chemical substances that are highly corrosive such as phenol and sulfuric acid will be handled during the experiment. Conclusion
Glycogen can be extracted from the liver tissues of animals using KOH solution and ethanol as the reagents. A calibration curve can be drawn by plotting the resulting absorbance values versus the standard glucose concentration. From here, the quantity of glucose in the commercial glycogen and the extracted glycogen are determined. The glucose content of commercial glycogen is 106 μg/ml while the extracted glycogen is 44 μg/ml. The purity of the extracted glycogen is calculated and the value is 0.21%. The low value could be a result from the inefficient drying process.
References
http://www.abcam.com/glycogen-assay-kit-ab65620.html [Accessed on 8th April 2014] http://www.nature.com/jid/journal/v37/n5/full/jid1961134a.html [Accessed on 8th April 2014]
