The experiment that was performed over two weeks served to examine how the amount of enzyme, amount of substrate, presence of inhibitor, or the magnitude of temperature changes the reaction rate between the enzyme and substrate present. The enzyme used in these experiments was peroxidase which was extracted from turnips and placed in flasks to be transferred into tubes and would later be mixed with the substrate to start the reaction. The substrate used in the experiment was hydrogen peroxide with the equation of H2O2 which reacted with peroxidase successfully (Wedig, 2019). The first two experiments served to test how increasing the amounts of the enzyme and substrate would affect the speed of the reaction rate between the two. The other two experiments performed on the week after tested the effects of an inhibitor, hydroxylamine, and the effects of different temperatures on the same reactions (Wedig, 2019). In all these experiments a substance called guaiacol was used to provide a visual clue to whether a reaction was occurring by turning a brownish color due to the presence of oxygen from the breakdown on the peroxide (Wedig, 2019). The amounts tested with the enzyme were .5, 1, and 2 mL which resulted in reaction rates of .0025, .0017, .0008 absorbance units per second respectively. The amounts of the substrate that were tested were .1,.2, and .4 mL which resulted in rates of .0037, .0012, .0008 absorbance units per second. As the enzyme concentration increased, more of the enzyme was able to bind with the substrate and increase the reaction rate. As the inhibitor was added, the reaction rate slowed down as the active sites are taken by the inhibitor hydroxylamine as it is a competitive inhibitor (Wedig, 2019).
Enzymes are catalysts that are catalysts for the chemical reaction that they were assembled for. Enzymes also can lower the amount of energy needed to start the reaction which is essentially just lowering the activation energy. By doing this it allows the cells within the body to conserve the energy that it can use for other cellular functions (Duncan, Johnson, Losos, Mason, Raven, 2019). For example, the human body can’t digest lactose as an energy source without using the enzyme lactase which enables the body to use it efficiently (Hopkins, 2019). This lab showed how changes within the environment of the enzyme alter how fast the reaction rate occurs, or even if the enzyme will function properly.
Materials and Methods
The enzyme used within the lab is the enzyme peroxidase. This enzyme was prepared by obtaining the extract of turnips which hold the amounts of peroxidase that were needed for the lab. The substrate, peroxide, is broken down by peroxidase throughout a series of reactions that oxidizes the peroxide and breaks down the peroxide into water. Guaiacol was used in this reaction as a color reagent which would indicate whether a reaction was occurring as the enzyme and substrate were mixed. When guaiacol was present in this reaction with peroxidase, it becomes oxidized, and as it becomes oxidized it changes from its colorless form and becomes a brown like color and becomes a substance known as tetraquaiacol. The spectrophotometer used in these series of labs measures in units of absorbance, which is directly proportional to reaction rate when absorbance is divided by time. The spectrophotometer measures absorbance by measuring how many photons are absorbed by the substance being placed into the spectrophotometer in a special container called a cuvette, and the number of photons absorbed depends on the wavelength or color of the light being shined through the substance examined. In this experiment, a light wavelength of 470 nm was used which corresponds to a light blueish light that was sent through the enzyme and substrate cuvette. To track the changes in absorbances, the absorbance of the solution was checked every twenty seconds in all labs up to 300 seconds total to provide a linear relationship between the data points. There will be no data from zero-twenty seconds as during that time the enzyme and substrate are mixed and then transferred into the cuvette which will then be inserted into the spectrophotometer to start the data collection at 20 seconds.
For the enzyme concentration activity, three different concentrations were tested with concentrations of .5 mL, 1 mL, and 2 mL. To perform this activity, first obtain a control group for low concentration containing 1mL of guaiacol, .5 mL of turnip extract, and 8.5 mL of water, for medium concentration use the same amount of guaiacol, but change the amount of turnip extract to 1mL and reduce the amount of water to 8 mL, and for high concentration change the amount of turnip extract to 2mL of turnip extract and use 7mL of water keeping guaiacol constant throughout. Then create the enzyme groups for the three concentrations using the same amount of turnip extract as the controls used for the respective concentration and fill the rest of the test tube with water up to 5mL. To create the substrates for each concentration, combine 1mL of guaiacol, .2mL of peroxide, and 3.8mL of distilled water. Then fill a cuvette with the control and set zero absorbance for the spectrophotometer then mix the enzyme and substrate and quickly transfer into the cuvette which is placed into the spectrophotometer. Once placed into the spectrophotometer, take absorbance measurements every twenty seconds starting at twenty seconds and ending at three minutes. Repeat these steps for the remaining concentrations and place the information into the table.
For the lab activity where the substrate concentrations were tested, .1, .2, and .4 mL of peroxide were tested to view the changes in absorbance over time. The three controls for this lab was created by mixing 1mL of guaiacol, 1mL of turnip extract, and 8mL of water. Then, starting with the low concentration substrate, mix 1mL of guaiacol with .1mL of peroxide and 3.9 mL of water. For medium concentration mix 1mL of guaiacol, .2mL peroxide, and 3.8 mL of water. For high substrate concentration, 1mL of guaiacol was mixed with .4 mL of peroxide with 3.6 mL of water. After preparing these substances, measure the absorbances using the same process as the enzyme concentration lab. Then create the enzyme tubes which will be mixed with the substrates by mixing 1mL of turnip extract with 4 mL of distilled water.
In the activity covering the effect of temperature on the reaction rate, four separate temperatures were tested. The temperatures used were 4, 23, 37, and 60 degrees Celsius. For each temperature create a control tube with 1mL of guaiacol, 1mL of turnip extract, and 8mL of water. To create the enzyme tube, mix 1mL turnip extract with 4mL of water. Finally, for the substrate, put 1mL guaiacol, .2 mL peroxide, and 3.8 mL water. Then place in the temperature that is being tested for at least 15-20 minutes before removing them and performing the measurements in the spectrophotometer.
Finally, the inhibitor that was used in this lab for the final activity was hydroxylamine, which was tested in amounts of 0, 1, and 5 drops of the competitive inhibitor which was added to the enzyme tube before mixing the substrate and the enzyme. To perform this experiment, a control containing 1mL guaiacol, 1mL turnip extract, and 8mL water with no hydroxylamine present. Then for the three amounts of inhibitor being tested, the enzyme tube is created with 1mL turnip extract and 4mL of water, and if inhibitor must be added then it should be added to the enzyme tube as to allow the inhibitor to properly mix for at least five minutes. Then create the substrate following the same process as the temperature activity. After those five minutes have passed, mix the tubes and follow the same process of measuring the change in absorbance over time.
Results and Discussion
As the enzyme concentration increased, the reaction rate increased as well just as it should be. In the activity it was shown that at low concentrations, the rate of the reaction was at only .0008 absorbance units per second and as it shifted to high concentration, the reaction rate increased up to .0025 absorbance units per second with the medium concentration having a rate of .0017 absorbance units per second. This increase is caused by the presence of more enzyme which causes more substrate to become broken down into water.
It shows how as the substrate concentration increases throughout the trials, the rate of reaction slows down as the solution will become more saturated with substrate than the amount of enzyme can handle. This effect is shown as the jump from low concentration to high concentration which is a jump from .0037 absorbance units per second to .0008 absorbance units per second which shows how peroxidase is overloaded by the increase in concentration of peroxide.
The effect that temperature has on reaction rate. In this activity, the enzyme being used works best at 37 degrees Celsius as peroxidase is found in humans and as a result operates best at human body temperature. This is shown by the data but however there was possible human error as 4 degrees Celsius appears to have the fastest reaction rate, but the enzyme should have denatured at this temperature and should have had a slower reaction rate.
The results were representative of the effect of an inhibitor, hydroxylamine, on the reaction rate. As the numbers of drops of hydroxylamine increased from 0 to 1 to 5, the reaction rate also decreased from .0017 to .0005 to .0002 absorbance units per second. This shows how the inhibitor effectively can compete with peroxidase for the active site.
- “Lab Module 5”. Dr. Cindy Wedig, 2019, pp. 4-5
- “Lab Module 6”. Dr. Cindy Wedig, 2019. pp. 5
- “Lactose Intolerance.” Johns Hopkins Medicine, www.hopkinsmedicine.org/health/conditions-and-diseases/lactose-intolerance.
- “Proteins: Molecules with Diverse Structures and Functions.” Biology, by Kenneth A. Mason et al., McGraw-Hill Education, 2019, pp. 46–47.