**B. Effect of Temperature on the Rate of Enzyme Reaction** ### a. Procedure 1. **Choose a concentration of amylase** that had an endpoint time of 60 to 90 seconds in Part A. This will likely be either 1.0 or 2.0 mg/ml. Only one amylase concentration will be used for this exercise. Pipette exactly 1 ml of this amylase concentration into each of 6 clean test tubes. 2. **Obtain 6 clean test tubes** and add 2 ml of starch solution and 2 ml of pH 7.0 buffer to each. You now have 6 identical test tubes with amylase and 6 identical test tubes with starch and buffer. 3. **Place a pair of test tubes** (an amylase test tube and a starch/buffer test tube) in each of the following temperatures: 100°C, 60°C, 45°C, 37°C, ice, and room temperature. Allow 10 minutes for samples to reach the correct temperature. 4. **Follow the timing procedure** in Part A and determine the endpoint times for each pair. To conserve time, if an endpoint has not been reached in 500 seconds, STOP, and record the end time as 500+ seconds. **Remember** to keep tubes in the heated or cooled bath water and return the contents of the dropper after each drop so that the temperature will not change during the experiment. 5. **Record all data** and answer questions below. ### b. Results 1. **Record your data below.** | Temperature (°C) | Endpoint Time in Seconds | |------------------|--------------------------| | 0 | 255 | | 25 | 60 | | 37 | 50 | | 45 | 150 | | 60 | 374+ | | 85 | 500+ | **Explanation:** This table shows how the temperature affects the rate of the enzyme reaction. As the temperature increases from 0°C to 37°C, the endpoint time decreases, indicating a faster reaction rate. Beyond this temperature, the reaction slows down, as shown by increased endpoint times at higher temperatures like 45°C, 60°C, and 85°C. **Exercise Instructions and Questions:** 2. Plot a graph comparing the temperature with the endpoint. The values of the independent variable (temperatures) should be plotted on the horizontal or X axis of the graph. The endpoints (values of the dependent variable, or data) should be plotted on the vertical or Y axis. c. **Questions** 1. What is the temperature optimum for this exercise? (At what temperature is the rate maximum?) *[Space for answer]* 2. Based on your data, predict what the endpoint would be at a temperature of 50°C. *[Space for answer]* 3. Why does the reaction go slower at temperatures cooler than the optimum? *[Space for answer]* 4. Why does the reaction go slower, or not happen at all, at temperatures much warmer than the optimum? *[Space for answer]* 5. During the exercise, what variables are held constant? What variables are manipulated? What variables are being measured? - Held constant? *[Space for answer]* - Manipulated? *[Space for answer]* - Measured? *[Space for answer]* 6. Predict what would happen if you boiled the enzyme tube before the reaction and then held the reaction at 37°C? Explain. *[Space for answer]* --- *[Note: This question sheet serves for educational purposes. Ensure to fill in the answers based on your experimental data and understanding of enzyme reactions and temperature effects.]*