MOHAWK COLLEGE OF APPLIED ARTS AND TECHNOLOGY CHEMICAL AND ENVIRONMENTAL TECHNOLOGY DEPARTMENT Lab Report ROOM NO: FE E309 EXPERIMENT NO : 9 TITLE : Factors Affecting Enzyme Function Submitted by Class Partners Instructor : Lyndsay Grover : BIOL 10000 lab. : Awatif Hagelamin : Farag Soliman Date lab performed : February 10, 2011 Date of submission : February 17, 2011 FENNELL CAMPUS HAMILTON, ONTARIO 1/4
Purpose: The purpose of this experiment was to observe and investigate the effects of ph on the function of an a-amylase enzyme. It is important for students of Biotechnology to understand the effects that ph can have on enzymes and their functions so to know the importance of ph stability in their work. Since every enzyme has an optimum ph and temperature and ph to work at, the concept of ph stability is important knowledge to have. Apparatus: - test tubes - vortex - beakers - pipette - graduated cylinder - spectrophotometer - Reagents: Maltose Colour Reagent, Concentrated a-amylase stock solution, 1% starch solution. Safety Guidelines: Wear gloves when handling the Maltose Colour Reagent (1% 3,5-dinitrosalicylic acid, 0.4 M NaOH, 1.06 M sodium potassium tartrate) as this concentration of NaOH is slightly corrosive. Flush your eyes with water if there is contact. Procedure: Enzyme Reaction Step 1. Label one test tube as blank and five others as ph5, ph6, ph7, ph8 and ph9. Add 1mL starch to each tube. To each assay tube add appropriate ph buffer, to the blank add ph7. 2. Make fresh dilution of a-amylase by mixing 0.1mL of concentrated stock solution with 9.4 ml of dh 2 O. Vortex to mix and keep stock and diluted enzyme on ice. 3. Add 1mL of dh 2 O to blank tube only. Cap and vortex. Add 1mL of dilute a-amylase solution to each of the other test tubes. Cap and vortex to mix. Incubate all six test tubes at room temperature for exactly 12 minutes. Maltose Detection Step 1. In the same order that starch solution was added, add 1 ml of the Maltose Colour Reagent to each of the tubes, including the blank. Cap, vortex and place tubes in a beaker of boiling water on a hot plate set at 100 C (boil exactly 15 minutes). Start timing when the Maltose Colour Reagent is added to first tube. 2. Place tubes on ice until cooled to room temperature. Add 9mL of dh 2 O to each tube. Tightly cap the tubes and invert several times to mix. 3. Transfer the solutions to Spec 20 tubes. 4. Using the blank zero the absorbance on the Spec 20 spectrophotometer. Measure the A 540nm of all the sample tubes. Record the absorbance of each sample in Table 1. 2/4
Data: Table 1 a-amylase as a Function of ph ph ph5 ph6 ph7 ph8 ph9 A 540 nm 1.198 0.919 1.044 1.149 0.994 Observations: - Starch solution was cloudy, no change after adding amylase solution, no change after vortex - Amylase solution was frozen, hard to break up, once thawed was a clear slightly beige colour - Changes after incubation period: none - Amylase was diluted with 9.4mL of dh 2 O instead of 9.9mL stated in laboratory manual - Changes after adding maltose colour reagent: solution is now a dark yellow colour - Colour change after heating: all phs turned a dark red brown - Colour change when adding distilled water: ph 9: dark orange-yellow, ph 8: dark yellow with a hint of orange, ph 7: dark yellow, ph 6: slightly lighter dark yellow, ph 5: medium yellow, blank: yellow. Discussion: This experiment was an interesting one to perform because it introduced us to some new elements that we have not used before. The idea that ph can have an effect on enzyme function is not a concept to which we have been properly introduced so to observe is first hand was an incredible learning experience. Also how with the use of the Maltose Colour Reagent we were able to determine enzyme activity from a spectrophotometer. Which is a function for the equipment that I wasn t aware was possible. The introduction of the new step in the procedure involving the vortex was a great introduction to a new piece of laboratory equipment and its function of rapidly mixing solutions. When looking at the observations and all the changes that occurred during the process you can really break down the steps in the reaction to observe enzyme function from the colour changes paired with the absorbance readings. Although the colour change does not correlate to the absorbance readings in the 3/4
way one would think, it is interesting to see how maybe the ph played apart into the colour change and not just the enzyme function. Sources of Error: Possible Sources of Human Error: Improper amount of ph buffer. Improper dilution of a-amylase. Solution not allowed to incubate for proper amount of time. Improper mixing. Improper amount of Maltose Colour Reagent added. Solution not heated for long enough. Improper dilution of solution with distilled water. Possible Sources of Equipment Error: Vortex working improperly. Hot plate not working properly or uneven heat distribution. Spectrophotometer not calibrated or calibrated improperly. Spectrophotometer set to wrong setting. Possible Sources of Experiment Error: Incorrect concentration of a-amylase solution. Incorrect concentration of Starch solution. Mislabeled ph bottles. Incorrect concentration of Maltose Colour Reagent. Post Laboratory Questions: 1. The absorbance ratings can be used to determine the enzyme activity at varying ph levels. After adding the ph buffer to each solution and heating it and then cooling it, we have added three different changes to the enzyme that may affect its ability to function. When the maltose is added it is meant to react with the enzyme and the colour change indicates its amount of activity. Due to the colour of the solution we can then use the spectrophotometer to read its absorbance levels. So in absorbance of the colour is related to the activity of the enzyme in the solution. 2. When regarding the graph it appears that ph 7 is the optimal ph for the a-amylase solution. The absorbance reading for ph7 occurs in the middle of all the other points and is likely close to the average of the absorbance reading. In consideration for the function of a biological component it would seem that a neutral ph would be the ideal ph for it to function at since a majority of biological components, especially in the human body, have a ph of 7. 3. In industries such as biopharmaceuticals the maintenance of ph and temperature are critical to enzyme function. The enzymes are needed to bind molecules to one another or to carry out a chemical reaction so their ability to function properly is critical to ensure the product is made. For this the company will need to create equipment to ensure that stable temperatures and levels of ph are maintained. Conclusion: Once the results for absorbance were plotted on the graph it was interesting to see the formation of the data. The wave like trend shown on the graph shows that ph and enzyme doesn t follow a linear trend line but more of a sinusoidal. Viewing this shows the enzymes are greatly affected by ph in the slightest amounts and it can completely change the activity of the enzyme. The optimum ph for the a-amylase seems to be ph 7 since it is the median in respect to both x and y-axis on the graph. The other absorbance readings appear to be variations of the optimum level. When observing the data of absorbance one must determine how it relates to ph activity. I am under the assumption that the higher absorbance levels mean greater enzyme activity. Since the maltose determined the colour change and the colour change would have resulted from the amount of enzymes 4/4
maltose was able to react with. Since colour change is what is read by the absorbance levels, it is safe to assume that the higher the absorbance levels, the greater amount of enzyme function. 5/4