Effect of pH and Temperature on Enzymes

Biological Catalysts: Enzymes

Authors: Kerr, A., Thom., W., Brennan., J.

Biological Significance: The significance of this experiment was to observe the influence of PH and temperature on the rate of a reaction with an enzyme. This is important to understand under what conditions an enzyme will still properly function.

Hypothesis: Extreme temperature and PH levels will slow down the rate of reaction with an enzyme.

Discussion/Conclusions:

Part I: (type discussion/conclusions concerning effect of temperature (including boiling) and pH on enzymes.)

In order to function properly, enzymes must be in an environment that has a certain temperature and pH. The first test done was the effect of temperature on the enzyme peroxidase. Each test tube was a certain temperature when the enzyme was added and put in the spectrophotometer. We came to the conclusion that peroxidase functions best between 23°C and 32°C because both gave the highest readings of about 0.13 absorbance after 120 seconds. The test tubes at 4°C, 48°C, and 23°C (boiled enzyme), all gave low absorbance readings of about 0.09 after 120 seconds. Due to the large difference in absorbance, we drew the conclusion that radically high and low temperatures do not allow enzymes to function properly.

We also tested the effects of pH on peroxidase. Four separate tests were done using a pH of 3, 5, 7, and 9. After running the samples through the spectrophotometer, it was determined that peroxidase is most efficient at a pH of 5, given the reading of 1.237 absorbance after 120 seconds. A pH of 7 is still a semi-conducive environment for peroxidase with a reading of 0.871 absorbance after 120 seconds. However, at pH 3 and 9 the absorbance readings were very low, concluding that enzymes can not function properly at very acidic or basic pH levels, the pH must be neutral at around 5 for the enzyme to catalyze reactions properly.

Our overall results were that enzyme activity is at its greatest when it’s at a temperature between 23°C and 32°C and a pH of 5. When enzymes are at a low or high temperature or pH the conformation of the enzyme is changed. Enzymes have an active site which bind to molecules in order to catalyse reactions. In radical conditions, the active site can be altered inhibiting molecules from binding to the enzyme, rendering it useless.

Part II: (type discussion/conclusions concerning identification of enzyme(s) in saliva – explain role of each cup (#1-5) and if your results allow you to make any conclusions on presence/absence of a salivary enzyme.)

Cup one was designed as a positive control for starch and the conclusion drawn based on the iodine test was that starch is present in popcorn. Cup two was a negative control for starch and after performing the benedict's test we concluded that there was no starch present in water. Cup three was also a negative control for starch which was proven that there was no starch in saliva after performing a benedict's test. Cup four was a negative control for the mechanical process of chewing and after performing the benedict's test it was proven that the mechanical process of chewing does not convert the starch in the popcorn to simple sugars. Cup five was designed as a test for simple sugars in saliva which was proven to be true after the benedict's test was positive for simple sugars present in the chewed popcorn after being exposed to salivary amylase in saliva. The combined results of all five cups allowed us to draw the conclusion that when the enzyme present in saliva, salivary amylase, is introduced to popcorn it breaks down the starch to simple sugars. The salivary enzyme was only present in saliva which was concluded by the presence of simple sugars in cup five but no simple sugars were detected when popcorn was only exposed to water.

Responses to Review Questions:

Offer an explanation of why we use refrigerators. In other words, explain why a partially eaten hamburger gets moldy after a week if left on the kitchen counter, but takes much, much longer to get moldy if kept in a refrigerator. Note that the temperature of most refrigerators is 4°C.

At lower temperatures like a fridge, seen by the data in our lab, the rate of activity of enzymes is the lowest, meaning that the breakdown of the food because of enzymes is the slowest, keeping it fresh longer.

Explain how elevated temperatures such as 100°C (boiling) affect enzyme activity.

Enzymes are proteins, when proteins are brought to to too high of temperatures, boiling, they denature, when a protein denatures it can no longer perform its function. This happens with the enzyme when it is boiled, it can no longer catalyze reactions, making the rate of enzyme activity zero or close to zero.

Based on the data collected during lab, what is the likely pH of a turnip cell? Explain your rationale for this conclusion.

A pH of 5, because the rate of enzyme activity was significantly higher at a pH of 5 than any other pH level that was tested. The pH level with the highest enzyme activity reflects the pH of the turnip as the enzyme would work the best at the pH level that it is made to live in.

What conclusion could be made if the popcorn plus water test resulted in the same Clinitest reaction products as that with the popcorn plus saliva?

If the popcorn would have tested positive for simple sugars in the beginning we could have concluded that popcorn naturally has simple sugars already broken down in it, or if both tested negative, we would know that saliva does not have enzymes that are able to break down starch into simple sugars.