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The Effect of Temperature and pH on Enzyme Activity

The purpose of our lab was to observe how the reaction rate of an enzyme increases or decreases when combined with different substrates. Enzymes are catalysts that increase the rate of biochemical reactions. The rate of these reactions can be affected by its environment: temperature and pH. Low temperatures can cause enzyme reaction rate to become inactive or very slow by causing less collision of molecules and high temperatures can cause enzymes to denature prompting the reaction rate to lessen. As temperature increases, the rate of an enzyme-catalyzed reaction should increase. Similarly, the pH of an environment can affect the shape of an enzyme decreasing or increasing its reaction. In addition, all enzymes have a different optimal pH. As pH increases, the rate of an enzyme-catalyzed reaction should increase. In this lab, to examine the effects of temperature and pH on the reaction rate of an enzyme, we utilized potato extract (catecholase) as our enzyme combined with the substrate catechol. We utilized the Spec 20 to determine the reaction rate of enzymes under different conditions because once the enzyme and substrate are combined in the test tube the reaction begins to occur forming benzoquinone. The product benzoquinone is a brown color and the darker the color the less light will be able to pass through the test tube causing the percentage of light absorbed to increase. The absorption of light will be measured by the Spec 20 allowing us to observe the enzymes reaction rate.
Potato Extract
Glass Tubes
This lab was a two-part lab where we analyzed the rate of enzyme reactions at different temperatures and later we looked at how pH levels are affected by enzyme-catalyzed reactions. To test the effects of temperature on enzymatic reactions, we filled 5 test tubes with 4 mL of water and 1 mL of potato extract. Then we filled another tube, which we referred to as the blank, with 1 mL of potato extract and 6ml of water (the extra 2 mL of water is added to offset the 2mL of catecholase that will be added later). From here we put all the tubes except the blank, which served as the control of the experiment, into different temperature environments. The temperatures used were 0℃, 18 ℃, 30 ℃, 40 ℃, and 60 ℃. We left the tubes in these conditions for 5 minutes before adding catechol to the tubes then we reinserted the tubes back in these temperature controlled environments for another 5 minutes. The next step after this was to test light absorbance through the use of the Photospectrometer (Spec 20). The higher the absorbance value determined the rate of the chemical reaction. For the section portion of the lab, we tried to see how pH balance affects enzymatic reaction rates. We filled the 5 tubes with 1 mL of potato extract and 4 mL of pH buffer solution. Each tube had varying pH buffer levels. The levels were pH levels of 3, 5, 7, 9, 11 and of course the blank which is our control tube was filled with 1 mL of potato extract and 4 mL of pH 7 buffer which is neutral. We then used parafilm and inverted to mix each solution and added 2 mL of catechol to all 5 test tubes. We waited 5 minutes for the chemical reaction to take place before calibrating the Spec 20 to test the absorbance.
Table 1: Effect of Temperature on Enzyme Reaction Rate
Temp (℃)
Absorbance after (5 minutes)
Graph 1: Effect of Temperature on Enzyme Reaction Rate

Table 2: Effect of pH on Enzyme Reaction Rate
Absorbance after (5 minutes)
Graph 2: Effect of pH on Enzyme Reaction Rate

As seen in table and graph 1, the effect of temperature on enzyme reaction rate, as the temperature increased the absorbance also increased until it peaked at sample 4 with a temperature of 40℃ which had an absorbance of .211. In the last sample, sample 5 with a temperature of 60℃, the absorbance began to decrease with an absorbance of .112. In table and graph 2, the effect of pH on enzyme reaction rate, the highly acidic samples (1 and 2) had greater absorbance than the basic samples (4 and 5) which had the lowest absorbance out of all the samples. The highest absorbance was in sample 3 (absorbance of .197) which had a neutral pH of 7.
DISCUSSION (need to be done)
Directions: This section should not just be a restatement of the results but should emphasize interpretation of the data, relating them to existing theory and knowledge. Suggestions for the improvement of techniques or experimental design may also be included here. In writing this section, you should explain the logic that allows you to accept or reject your original hypotheses. Provide a conclusion based on the results that you got.
example: My hypothesis was clearly false, as some ions or cofactors had more rates of absorbency than others. This data suggested that copper is the most crucial to the enzymatic reaction as it had the lowest absorbency when bound to prevent reaction. Cofactors such as copper, calcium, and magnesium were tested by using chelating agents to bind them to prevent them from catalyzing them enzymatic reaction. As a result, their efficiency entirely depended on low absorbance rate. Since Tube 4 was the control, we would have thought that it would have the highest absorbance rate as the enzymatic reaction there were no chelating agents binding it.This may have been a possible error such as miscalculation on the portion of the mixture such as having lest EDTA chelating agents, that Tube 1 had fingerprints.There may also be possible errors such as
inaccurate timing mixing the catechol into the solution or the water bath. If this is not the case, we would suspect that there are cofactors of the enzyme that could actually slow down the enzymatic reaction rather than speeds it up. The rate of absorbance was calculated by subtracting the absorbency at 20 minutes to the absorbency at 10 minutes. I concluded this experiment may have possible errors, but suggests that copper bound by Citric acid is the most effective for the
enzymatic reaction of benzoquinone.


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