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# UCSD Benedict Test on Known Solutions Deionized Water Enzyme Activity Worksheet

Question 1. Any enzyme converts substances generally known as substrates into substances commonly called products. For the reaction shown above, name the substrates and products below. Then label them on the drawing above.

1. substrate(s):
1. product(s):

Table 1: Benedict’s Test on Known Solutions

 Test Tube Number Test Solution Benedict’s Test Results: Color Benedict’s Test Results: Precipitate (-, +, ++, +++, etc.) 1 1% sucrose 2 1% fructose 3 1% glucose 4 DI* water

* deionized water (water which contains no ions)

Question 3. What was the purpose of testing deionized water?

Question 4. A student arrives at the lab on Saturday. S/he wants to verify that the stock invertase solution left over from Friday’s lab is still “good”. The student mixes some 1% sucrose and some of the stock enzyme solution in a test tube and lets it stand in the test tube rack for one-half hour. Then the student performs a Benedict’s test on the test tube contents.

1. What is the correct interpretation of the Benedict’s test if the result is a clear, blue solution? Is the enzyme active or not?
1. What is the correct interpretation if Benedict’s test shows a bright orange precipitate? Is the stock enzyme still “good”?

Question 5. Based on what you already know about living things, in what general range of temperature do you expect enzyme molecules to function best? We will have 4 “groups.”

1. What is the dependent variable?
1. What is the independent variable?
1. State your answer as a hypothesis for this part of the exercise in an If…, and …, then … format.

Table 2: Invertase Activity at Different Temperatures

 Test Tube Number Temperature of Test Solution Benedict’s Test Results: Color Benedict’s Test Results: Precipitate (-, +, ++, +++, etc.) 1 0 o C 2 25 o C 3 40 o C 4 100 o C

Question 6. At which temperature was invertase most active?

At which temperature was invertase least active?

Describe how you arrived at your decision:

Question 7. Recall Exercise 3: “Diffusion and Osmosis”, where you observed the India ink dye. What happened to the molecular motion as you increased the light intensity (heat)? In that exercise, you learned the relationship between heat and molecular motion.

1. State the relationship:
1. Referring to Table 2, identify which of your results can be explained by this relationship. Explain your reasoning.
1. Which part of your Table 2 data are not consistent with the relationship between heat and molecular motion?
1. What do you know about enzymes (and proteins in general) which can account for your results at 100°C? (You may need to consult your textbook’s discussion of protein molecules to answer this question.) This should include the following concepts:
• Structure/function/denaturation
• Bonds – types of bonds affected

Question 8. List the pH’s of the above buffered solutions in order from most acidic to most basic:

(most acidic) (most basic)

Question 9. What pH is called “neutral”? ______________

Question 10. What is the pH of pure water? ______________

Question 11. Based on what you already know about living things, in what general range of pH do you expect enzyme molecules to function best? We will have 4 “groups.”

1. What is the dependent variable?
1. What is the independent variable?
1. State your answer as a hypothesis for this part of the exercise in an If…, and …, then … format.
2. TABLE 3: INVERTASE ACTIVITY AT DIFFERENT pHs
 Test Tube Number pH of Test Solution Benedict’s Test Results: Color Benedict’s Test Results: Precipitate (-, +, ++, +++, etc.) 1 1.0 2 4.4 3 8.0 4 12.0

Question 12. At which pH was invertase most active? _______________

At which pH was invertase least active? _______________

Question 13. Thought question: Assuming yeast cells (from which your invertase was extracted) could survive pH 1.0 or pH 12.0, do your results suggest that they could obtain their energy from sucrose? Explain.

Question 14. In the body of an organism such as yourself, do you expect all enzymes to operate effectively in the same pH range? For example: compare the pH of the stomach, where the digestive enzyme pepsin works, and the pH of blood, where other enzymes are found.

What would you guess to be the normal pH inside of human cells, where there are many different enzymes?

Question 15.

1. Name the enzyme to be used in your experiment:
1. Name the substrate to be used in your experiment:

Question 16. What is the dependent variable of this experiment?

Question 17. What is the independent (experimental) variable of this experiment?

Question 19. Describe what is meant by the phrase “controlled experiment.”

Question 20. Your instructor suggests that the total volumes in all tubes should be identical. Why?

Question 21. What is the solvent in all of your solutions?

How can the solvent be useful in designing your experiment?

(Hint: See Question 19.)

Question 22. Some students decided to use the same pH buffer solution in all of their tubes. Why would this be useful? (Which buffer solution will you use? Why? (Hint: Review your results in Table 2.)

Question 23. What temperature will you use for your experiment? Why? (Hint: Review your results in Table 2.)

Question 24. Students have suggested that the enzyme be “pretreated” with the iodine before adding the substrate. Why would this be useful? (Hint: If you added the substrate to the enzyme first, think what might be happening while you were on your way over to the iodine bottle.)

Question 25. How much time will you allow for the enzyme-substrate interaction before performing Benedict’s test?

Table 4: Experimental Design for

Effect of Iodine on Invertase Activity

(Note: the questions on pageS 14-15

will tell you how to complete this table)

 Solutions Tube # 1 Contents Tube # 2 Contents Tube # 3 Contents ml. (or “drops”) of iodine 0 ml 1 ml 3 ml ml. enzyme (name: ________________) 2 ml 2 ml 2 ml ml. substrate (name: _______________) 2 ml 2 ml 2 ml ml. buffer (pH: _______________) 4 ml 4 ml 4 ml ml. DI water Total Volume

Question 26. Results: execute your experiment as planned, then perform Benedict’s test and enter your data in the table below.

 Tube # 1 Tube # 2 Tube # 3 Benedict’s test results

Question 27. Iodine experiment conclusions: