Acid and Enzymatic Hydrolysis of Glycogen - Lab Report Example

The acid and enzymatic hydrolysis of glycogen Introduction: Polysaccharides are the high molecular weight molecules formed by joining the monosaccharide units using the glycosidic bonds. Some important polysaccharides are starch, cellulose and glycogen. They are the polymers of the glucose. This is proved by the acid hydrolysis of these polysaccharides, which yields the monomer glucose molecules. Partial hydrolysis of the glycogen yields maltose and dextrans, indicating that the alpha glycosidic bonds are between the C-1 and C-4 carbon of the adjacent glucose molecules in the maltose and the C-1 and C-6 carbon of the adjacent glucose molecules in the dextrans. Glycogen structure is similar to the amylopectin molecule and it is highly branched. The presence of the monomer compounds are identified by both acid and enzymatic hydrolysis. The acid hydrolysis takes place over a period of time. The acid hydrolysis by the mineral acids takes place readily. The acid hydrolysis product is glucose.(Melville and Alsberg 1930). The enzymatic hydrolysis of the glycogen by the a-amylase (a(1® 4) glucan, 4-glucanohydrolase, E.C.3.2.1.1) cleaves the alpha 1 4 linkage in the glycogen molecule yielding a mixture of glucose, maltose and dextrin at the end. (Barbour, 1929). The alpha 1 6 hydrolysis does not takes place as the enzyme is specific for the alpha 1 4 cleavage. As the hydrolysis occurs in a random manner, a variety of the products are formed. (Plummer, 2001).In both the hydrolysis procedures the end product differs. The glucose is the only compound in acid hydrolysis whereas in the enzymatic hydrolysis glucose, maltose and dextrin are the products. The end products are reducing sugars hence the estimation of the reducing sugars is carried out by the Dinitro salicylic acid method. The increase in the number of the reducing sugar production as the hydrolysis takes place is determined by the Dinitro salicylic acid method. The reducing sugars have free carbonyl group with them. This free carbonyl group is oxidized by the 3,5-dinitrosalicylic acid (DNS). Simultaneously the DNS is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions. This is a basic redox reaction. (Miller, 1959). Oxidation step: Aldehyde Group --- carbonyl group Reduction step: 3, 5-dinitrosalicylic acid -- 3-amino,5-nitrosalicylic acid. The reducing sugars are easily measured by the 3,5-dinitrosalicylic acid (DNS) method. According to the Lambert Beer’s law, the concentration of the solution is directly proportional to its optical density. So if the absorbing index of a particular solution is known then the concentration of the given solution at that particular wavelength can be determined by measuring the optical density of the solution at that particular wavelength. Thus one molecule of the sugar is reduced by one molecule of the DNS. This is the convenient, relatively simplest method of estimation of the reducing sugar. The only draw back of this method is the low level of accuracy of the results. Though it is a straight reaction, it is expected that many side reactions occurs such that the actual reaction stoichiometry is more complicated and varies for different sugars. The absorption is high at 540 nm. (Passonneau and Lowry 1993). Both the acid and enzyme hydrolysis are carried out in the same time in order to compare and estimate the glucose production. The main aim of this experiment is to compare and estimate the rate of production of the glucose in the acid hydrolysis and the enzymatic cleavage of the glycogen. Materials Required: 1. 1.2 M NaOH 2. 2M HCl 3. 3, 5, Dinitrosalicylic acid reagent solution ( Dintirosalicylic acid 10 g , phenol 2 g, sodium sulfite 0.5 g , sodium hydroxide 10 g is dissolved in 1 liter of the water to prepare 1% DNS solution ). 4. Rat liver Glycogen in water 5. Rat liver glycogen in phosphate buffer. 6. Amylase ( ph 6.9) Methods: Acid Hydrolysis: The 0.4 cm3 of the glycogen solution, having a concentration of 8mg cm-3 in water was prepared. To this solution, 0.6 cm3 of the 2M HCl is added. Ten test tubes were prepared with the following solution and kept in a boiling water bath for the complete hydrolysis to take place. The reaction was stopped at various time intervals by the addition of the 1.2M NaOH. This strong base will neutralize all the acid present in the solution and as a result there will be no acid for the hydrolysis and the reaction was stopped. (Bancroft and Fry 1932). The reactions were stopped at the various time intervals and kept aside. To the test tubes 0.5 cm3 of the 3,5-dinitrosalicylic acid and it was heated for 5 minutes in a water bath. The development of the blue color indicates the concentration of the glucose molecule in the solution. After incubation for 5 minutes the solution was cooled and 8.5 cm3 of water is added and the intensity was measured at 540 nm. Enzymatic hydrolysis: In the enzymatic method of hydrolysis the 0.4 cm3 of the glycogen solution, having a concentration of 8mg cm-3 in water was prepared. To this solution, 0.6 cm3 of the amylase solution was added. The Ten test tubes were prepared with the above solution and kept at room temperature to allow for the hydrolysis reaction to occur. The reaction was stopped at various time intervals by the addition of the 1 cm3 of the 3,5-dinitrosalicylic acid. The solution was then kept at the boiling water bath for 5 minutes for the development of the color. The intensity of the color indicates the concentration of the product in the solution. After cooling the solution, 9 cm3 of water was added and then the color intensity was noted at 540 nm. Results: Graph 1: Estimation of glucose by acid hydrolysis method: Table 1: Estimation of glucose by Acid hydrolysis: Time (minutes) Absorbance 540 nm Concentration ( g/l) 0 0 0.00 1.5 0.778 4.88 3 0.977 6.13 4.5 1.176 7.37 6 1.312 8.23 9 1.548 9.71 12 1.692 10.61 15 1.704 10.68 20 2.26 14.17 25 1.948 12.21 The table 1 indicates the glucose estimation values for the acid hydrolysis method. The graph 1 indicates the concentration values of the solutions at different time intervals. It is found from the graph that the maximum concentration was at 20th minute. Graph 2: Glucose estimation by enzyme hydrolysis method: Table 2: Glucose estimation by Enzyme hydrolysis method: Time (minutes) Absorbance 540 nm Concentration ( g/l) 0 0 0.00 1.5 0.134 0.84 3 0.257 1.61 4.5 0.358 2.24 6 0.555 3.48 9 0.665 4.17 12 0.797 5.00 15 0.841 5.27 20 0.889 5.57 25 0.92 5.77 The table 2 indicates the concentration of the glucose at various time intervals. It was found that the concentration was higher as the time interval increases gradually. Discussion: Acid hydrolysis: The acid hydrolysis produced more amount of Glucose than the enzyme hydrolysis. The concentration of the glucose was found to increase gradually with respect to the time. The concentration of the glucose at the 1.5, 3, 4.5, 6, 9, 12, 15, 20, 25 minutes were 4.88, 6.13, 7.37, 8.23, 9.71, 10.61, 10.68, 14.17 and 12.21 g/l. At the 12th and 15th minute the concentration of the glucose was found to be almost the same and then the increase was markedly good. The concentration of the glucose was found to be maximum for the 20th minute and after which the concentration of the glucose was found to be lower. At the 25th minute the concentration was 12.21 g/l when compared to the 20th minute where the concentration was 14.17g/l. The concentration of the glucose at 3 minutes was 6.13 g/l. This increased up to a maximum of 14.17 g/l. Enzyme hydrolysis: The enzyme hydrolysis was a very slow process when compared to the acid hydrolysis. The yield was also very less. The concentration of the glucose at the 1.5, 3, 4.5, 6, 9, 12, 15, 20, 25 minutes were 0.84, 1.61, 2.24, 3.48, 4.17, 5, 5.57, 5.77 g/l respectively. The initial concentration of the glucose at the 3rd minute was 1.61 g/l only. As the time increased from 0 to 25 th minute the concentration of the glucose in the solution also increased. The increase in the concentration of the glucose in the solution did not come to an end. So even if the solution is kept for some more time the concentration of the glucose might have increased. Conclusion: It was found from the experiment that the acid hydrolysis yielded more amount of the glucose than the enzyme hydrolysis and the rate of product formation was also found to be higher for the acid hydrolysis method. The amount of glucose production was higher in the acid hydrolysis method and whereas in the enzyme hydrolysis along with the glucose the maltose and little amount of dextrin were also formed. The conversion of the maltose into glucose should occur in the enzyme hydrolysis method and this delay the rate. References: Bancroft, G and Fry, EG 1932, Adsorption and Hydrolysis of Glycogen, The Journal of Biological Chemistry, Vol.100, pp.255-265. Barbour, AD 1929, Enzymatic hydrolysis of Glycogen, Journal of Biological Chemistry, Vol. 85, pp.29-45. Melville, S and Alsberg, CL 1930, The hydrolysis of glycogen in various concentrations of acids, and the hydrolysis of glycogen with takadiastase, The Journal of Biological Chemistry, Vol. 93, no.2, pp.235-254 Miller GL. 1959, Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar, Analytical Chemistry, Vol.31, no.3, pp.426–428. Passonneau, JV and Lowry, OH 1993, Enzymatic analysis: a practical guide, Springer publications. Plummer, DT 2001, An Introduction to Practical Biochemistry, Tata McGraw- Hill publications. Read More