Assignment 2: pH Optimum for Invertase

Another factor that strongly influences enzyme activity in living cells is the pH of the environment in which the enzyme is designed to function. For example, in humans, a cytoplasmic protein in a skin cell is surrounded by a different fluid environment at a different pH than a membrane-bound enzyme like invertase with an active site that projects out into the lumen of the small intestine. The following exercise is designed to help you understand the effect of pH on enzyme activity by studying invertase activity over a range of different pH values from acidic to basic conditions.

Formulate a hypothesis to predict the effect of pH on invertase activity.

Set up an experiment at the optimal temperature that you determined in assignment 12, with a substrate concentration of 90 mM. Beginning at the lowest pH value, 3.0, measure invertase activity, find the slope of the line, record data, and repeat this process for other buffer solutions with different pH values. Run experiments for at least two different buffers for each whole number change in pH units until you reach the maximum pH value of 10.0 (e.g., 3.0, 3.4, 4.0, 4.4, 5.0, 5.4).

Create a plot of V_O vs. pH. Click on this plot and drag the gray dashed pH line until you find V_max. When this line is correctly aligned, you will have found the pH optimum for invertase under these reaction conditions. The optimal pH value will appear in the best pH text box.

What is the optimal pH for invertase activity? Do the results of this experiment support or refute your hypothesis? Why or why not? Explain your answers? Why and how do pH changes affect invertase activity?

If you were to carry out these pH experiments at a higher or lower temperature, what effect would this have on the pH optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results.