Purpose: To examine how an acidic fluid will modify the outcome of an enzymatic reaction.
Introduction: Enzymes are “specialized proteins made by living cells that serve as biological catalysts.” (eScience Labs, LLC, 2013) These enzymes “allow certain industrial processes to be carried out at normal temperatures and pressures, thereby reducing the amount of energy and expensive equipment needed.”(BBC, 2012) Enzymatic activity can be affected by three factors, pH, salt, and temperature. In this project, we will be seeing how acidic fluids will affect the enzymatic reactions. Furthermore, this experiment will answer the following questions: What level of pH there must be in order for the reaction to slow down, or speed up? Are there specific levels of ph levels that speed up enzymes? Seeing that enzymes tend to do great on themselves, my hypothesis is that acid fluids (low pH levels) will actually hinder the enzymatic reaction. My reasoning behind this is that the substrate will take a longer time to mix with the fluid acids, and thus much of the time will be wasted when the enzyme makes contact with the combined substances.
In order to do this, we will be using the following materials: Yeast Packet (this will be our enzyme, which comes from (source)), 30 mL 3% Hydrogen Peroxide (this will be our substrate), (2) 250 mL Beakers, 3 Balloons, H 2 O 2, Measuring Spoon, Permanent Marker, Ruler, 30 cm String, 3 Test Tubes (Glass), Test Tube Rack, Stopwatch, 3 Acidic Solutions (Orange juice, 4.5% Acetic Acid (Vinegar), C 2 H 4 O, and saliva). For us to be able to examine how an acidic fluid will modify the outcome of an enzymatic reaction, we will need to measure and see how fast the reaction takes place, and how big the reaction was. Thus, in order for us to do this, we will:
1. Use a permanent marker to label test tubes 1, 2, 3, and 4. Place them in the test tube rack.
2. Fill each tube with 10 mL hydrogen peroxide.
3. Fill test tube 1 with acidic fluid 7 ml of orange juice (pH = 3), test tube 2 with 7 ml of vinegar (pH = 2), and test tube 3 with 7 ml of saliva (pH = 10), leave test tube 4 with no acidic fluid (this will be your control). Swirl each tube to mix, and wait 30 seconds.
4. Meanwhile, find one of the balloons, and the piece of string. Wrap the string around the uninflated balloon and measure the length of the string with the ruler. Record the measurement in Table 1.
5. During the 5 minutes prepare the balloons with yeast by adding 1 / 4 tsp. of yeast each balloon. Make sure all the yeast gets settled to the bulb of the balloon and not caught in the neck. Be sure not spill yeast while handling the balloons.
6. Carefully stretch the neck of the balloon to help ensure it does not rip when stretched over the opening of the test tube.
7. After we have placed the 10ml of Hydrogen Peroxide in each test tube, and have also mixed in the acidic fluids, we will attach the neck of a balloon that we prepared to the top of each test tubes making sure to not let the yeast spill into the test tube yet. Once the balloon is securely attached to the test tube we will lift the balloon and allow the yeast to enter the test tube. Tap the bulb of the balloon to ensure all the yeast falls into the tube.
8. As soon as the yeast is starting to make contact with the hydrogen-acidic fluid mix have your lab partner (or a friend) start recording the time in which the enzymatic reaction is occurring. Record the following in Table 1.
9. Wrap the string around the center of each balloon to measure the circumference. Measure the length of string with a ruler.
What effect does the acidic treatment have on the enzyme activity? As we see the results, we can determine that my hypothesis was wrong; it was not valid. The results showed that the closer the pH was to the optimum pH level of the yeast enzymatic, the faster the enzymatic reaction was. As we can see, the optimum pH levels of yeast enzymatic is of a pH of 7. When we recorded the reaction time of the saliva (pH of 10), it was a lot faster than that of the vinegar fluid, which contained a lower pH of 2. Thus, the effect that the acidic treatment had on the enzyme activity depended on how close it was to the optimum pH levels of the enzyme. This does not mean that the pH is 7 for all types of enzymes, but that each enzyme has it’s own optimum pH level, and the closest the pH to the optimum level, the faster the enzymatic reaction occurs. Looking back, how could you have improved your experiment?
Looking back, I could have improved my experiment by adding more test tubes and using more different pH levels, so it would be easily seen that a pH of 7 would have almost an immediate reaction, while a pH of 5-6 or 8-9 would be slower- this would directly clarify and precisely prove the experiment. What is your conclusion? Was your hypothesis supported?
My conclusion is that the closer the pH of the acid is to the optimum pH of the enzyme, the faster the enzymatic reaction is. This means that “Changes in pH also alter an enzyme’s shape. Different enzymes work best at different pH values. The optimum pH for an enzyme depends on where it normally works.”(BBC, 2012) To further explain this, here is a line graph that clearly explains the enzyme activity against pH.
Here we can see that the optimum pH for the yeast is 7 (blue dotted vertical line), and that the red line signals the pH levels. As we can see, the closer the pH gets to the optimum pH, the faster the enzyme activity is, and the further it is, the slower the activity becomes. Furthermore, my hypothesis was not supported, because as stated earlier, acidic fluids will make the reaction go faster or slower depending on how close they are to the enzyme’s optimum level. To answer my introduction questions, “What level of pH there must be in order for the reaction to slow down, or speed up?” The level of pH that there must be in order for the reaction to slow down, or speed up depends on how close it is to the optimum pH of that enzyme. “Are there specific levels of pH levels that speed up enzymes?” No, there are not specific levels of pH that will speed up any enzyme. “Changes in pH alter the state of ionization of charged amino acids that may play a crucial role in substrate binding and/or the catalytic action itself.” (RCN, 2014)
EScience Labs LLC. (2013). Custome Lab Manual: UMUC BIOL 102/103. 12 Chalet-London, MD: eScience Labs LLC: More Than An Experiment, An Experience. Retrieved July 24, 2014. BBC. (2012, June 12). BBC – GCSE Bitesize: Temperature, pH and enzymes. Retrieved July 24, 2014, from http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/proteins/proteinsrev3.shtml RCN. (2014, April 18). Enzymes. Retrieved July 24, 2014, from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html