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Atılım University School of Medicine
MED103 Medical Biochemistry
EXPERIMENT 2: ENZYME KINETICS
Name: Ahmed Al-Adilee ID: 19261084003 Group: C
Objectives:
The aim of this experiment is to understand and observe the activity of the enzyme amylase, and to detect the presence of starch in a sample.
Introduction:
Complex carbohydrates and sugars are related molecules. The chemical structures of all the carbohydrates, in fact, are related. For example, starch is a polysaccharide consisting of many glucose units connected together. The polysaccharides cellulose (from plants) and glycogen (from animals) are just very large molecules made by connecting glucose rings in different ways basically. The starch (amylose) molecule shown above contain α-1,4 glyosidic bonds, which are hydrolyzed during digestion by the action of the enzymes in saliva and in the small intestine. enzymes are basically biological catalysts that speed up or increase the rate of the metabolic reactions, they achieve that by reducing the activation energy of a reaction without altering the equilibrium constant. This group of enzymes are called amylases and they hydrolyze the starch. They hydrolyze polysaccharides like glycogen and starch in our digestive system. The pancreas
and salivary gland make α-amylase to hydrolyze dietary starch into disaccharides and oligosaccharides which are then converted by other enzymes to glucose to supply the 2 body with energy. Starch consists of two forms which are amylose and amylopectin. Amylose consists of a linear helical polymer with α (1-4) glyosidic bond while amylopectin is a branched polymer containing glucose with α (1-4) glyosidic bond and α (1-6) glyosidic bonds at branch points as shown below. We will detect the presence of starch in solution using iodine solution as an indicator. Iodine (I 2 ) is a deep blue/dark blue in the presence of starch, Iodine forms a large complex with amylose helix giving a blue color. As starch is broken up to dextrins, the iodine turns to a brown/red color, followed by a pale brown/yellow when the enzyme amylase has completed hydrolysis which at this point the starch loses its ability to stain with iodine. It's important to understand here that the enzyme amylase, and most of the other enzymes generally, are globular proteins (tertiary structure), they are affected by pH, temperature, organic solvents, concentrated urea solution, inorganic salts and many other denaturing agents which is a process in which the protein molecule losses its structure/shape due to external stress and thus losses its function and property of forming enzyme-substrate complex and become in active.
Observation: Saliva will be used as a source of amylase in this experiment. The enzyme
activity can be observed via the degradation of starch. Effects of the temperature and pH on enzyme activity will also be evaluated.
- Measure the pH of the sample in the first tube (Tube is named as 1) by an indicator paper and compare it to the pH value table and note the value. This sample will be used as a control group (as the sample containing the active enzyme).
- Add 4-5 drops of 1 M HCl to equalize the pH of the sample in the second tube to 1 (Tube is named as 2) for use as an enzyme sample denatured with an acid.
- Keep the sample in the third tube (Tube is named as 3) in the boiling water bath for 10 minutes (for use as an enzyme sample denatured with temperature). Repeat the following steps for all samples in test tubes. Temperature was at 60 C.
- Add 1 ml of 1% starch solution is to a 3 new test tubes (Tubes are named as S). Put 5- 6 drops of iodine solution into the first tube and observe the color change. Meanwhile, be sure that a watch is ready to use.
- Pipette 2-3 drops of enzyme sample the first test tube. Begin to stir the tube and start the time simultaneously. Record the color in the tube at t=0. Keep time until you see a brown-red color in the tube and record the time.
- Repeat steps 8-10 for second and third tubes. Keep the same time found for the first tube and note whether the color change is observed. Do not forget to stir the tube during the process. Discuss all findings of the experiment with your teammate and write and discuss them in your lab report. We made sure during the experiment to use new Pasteur pipettes for each solution and for each chemical transfer separately in order not to mix solutions and this was to reduce possible sources of errors that might affect our expectations of the reaction and as I will state later in the discussion part of this report. I noted the changes in the test samples, the first one (the control group) we measured the pH by using an indicator paper and it had pH value of 7, for the second test tube we examine pH change effects on the enzyme as we added HCL to equalize it to 1, while in the third we examine temperature changes, placed the test sample in the water bath for 10min at 60°C.
Experiment task table:
1 Estimated pH of the first tube (T1) pH= 2 Color of the first tube at t=0 Dark-blue 3 Time (min) when brown red color is observed in the first tube
4 Color of the second tube (T2) at t=0 Dark blue 5 Did you observe a color change in the second tube when you kept the same time? No 6 Color of the third tube (T3) at t=0 Dark blue 7 Did you observe a color change in the third tube when you kept the same time? No
Discussion:
In this part firstly I want to discuss the results I noted in my table, the estimated pH of the first tube was neutral (pH=7) because this is the perfect pH value for the enzyme amylase to work in and the first test tube was our control group/sample from saliva, the enzyme is also secreted in the pancreas and its optimum pH is between 6.7-7.0. As for the color of the solution at the initial time was blue as it indicated that starch solution stained with iodine and as hydrolysis takes place the color changes degradedly to brown-red color, I want to point to the fact that it took a long time for the color to be observed in the sample because we might have increased the amount of the starch solution of approximately ±0.3ml which creates a margin of error that reduce accuracy when we try to compare and study the other test tubes treated with pH and temperature changes. To add to that it is known that our classmate shouldn't eat, drink, smoke, or eat gum for 30 min before beginning collection of saliva, and probably he did all of that. I also observed that his saliva sample took a long time to filtrate as well and that may explain why did it take that much time for the color to appear in the samples. For test tube 2 the color was observes dark blue for the same reason mentioned previously, but this time no change in color was observed this is due to the fact that the enzyme when faced with large changes in pH values its denatures and losses its ability to bind to the substrate and thus cant hydrolyze starch which stained with iodine still and formed the absorbed blue color. Same thing with temperature (T3) and in this step, we used a water bath which is extremely helpful in reducing the margin of error as it is reliable and the temperature degree is digital and can be kept constant. Another one was the mixer or Vortex genie machine which helps solutions to be mixed constantly.
