Anatomy and Physiology I Fall 2000
Lab Session 4

Enzyme Action


Name _________________________
File # _______


Introduction
Enzymes are biological catalysts, functioning to increase the rate of chemical reactions. Most enzymes are proteins, and their catalytic action results from their complex structure. The great diversity of protein structure allows enzyme action to be highly specific. The enzyme salivary amylase is present in human saliva. Amylase hydrolyzes starch (a polysaccharide) into maltose, a sugar (dissacharide). This enzyme will be used to demonstrate the effect of temperature and enzyme concentration on enzyme activity. Before coming to lab make sure to read Chapter 4 pp. 79-84 "Enzymes as Catalyst and Control of Enzyme Activity."



LAB PROCEDURE

I. The Effect Of Temperature on the Rate of Action of Salivary Amylase

1. Label three beakers as follows:
1) ice water
2) room temperature
3) body temperature

2. Pour approx. 10 ml. of a 1% starch solution into each of the three beakers.
Place beaker 1 into an ice water bath and record the temperature of the ice water.
Leave beaker 2 at room temperature.
Place beaker 3 at 37 C.
Make sure that these three beakers stay at their respective temperatures throughout the whole investigation.

3. Test each of the starch solutions in beakers 1, 2, and 3 for the presence of starch and for the presence of sugar as outlined below:

A. Iodine Test
On a petri plate or glass slide at room temperature, mix one drop of solution with one drop of iodine. Observe and record color of solution in the table provided on p. 2.
Black/Purple color reaction = starch is present
Light Brown/Yellow color (no color reaction) = starch is absent

B. Benedicts Test
In a test tube mix approximately 1 ml. solution with 1 ml. Benedicts solution. Place in a boiling water bath for 1 min. Immediately observe and record color of solution in the table provided on p. 2.
Red color reaction = high concentration of sugar
Orange/Yellow color reaction = medium concentration of sugar
Green color reaction = low concentration of sugar
Blue color (no color reaction) = sugar absent

4. Now add 1 ml. of salivary amylase solution to each beaker 1, 2, and 3, mix well, and record the time.

5. At 10 min. intervals after the salivary amylase was added, test each of the solutions in the beakers for the presence of starch (Iodine Test p. 1) and for the presence of sugars (Benedicts Test p. 1) as described in step 3.
Mix well prior to taking samples of solution for testing.
Do this for 10 min., 20 min., 30 min., and 40 min. after the addition of 1 ml. salivary amylase.
Observe and record color of solution in the table provided below.




Quantify the results recorded above as follows. Include assigned numbers along with the observed color reaction in the table above:

A. Iodine Test
Black/Purple color reaction = starch is present = 1
Light Brown/Yellow color (no color reaction) = starch is absent = 2

B. Benedicts Test
Red color reaction = high concentration of sugar = 1
Orange/Yellow color reaction = medium concentration of sugar = 2
Green color reaction = low concentration of sugar = 3
Blue color (no color reaction) = sugar absent = 4






II. The Effect Of Enzyme Concentration on the Rate of Action of Salivary Amylase


1. Label four test tubes as follows:
1) 1:10
2) 1:100
3) 1:1000
4) 1:10,000

2. Pour 9 ml. of distilled water into each of the four test tubes.

3. Add 1 ml. of salivary amylase to the first tube. Mix thoroughly. This gives a one to ten dilution (1:10).

4. Take 1 ml. of the 1:10 dilution and transfer it to the second test tube and mix (this is a 1:100 dilution).
Take 1 ml. of the 1:100 dilution and transfer it to the second test tube and mix (this is a 1:1000 dilution).
Take 1 ml. of the 1:1000 dilution and transfer it to the second test tube and mix (this is a 1:10,000 dilution).
Take 1 ml. of the 1:10,000 dilution and discard it. The test tubes should now hold 9 ml. each.

5. Place these test tubes at 37 C for 10 minutes.

6. At the end of the 10 min. incubation period briefly remove the test tubes from 37 C and place 1 ml. of the 1% starch solution in each test tube. Return the test tubes to 37 C.

7. At 10 min. intervals after the starch solution was added, test each of the solutions in the test tubes for the presence of sugar using the Benedicts Test on p. 1.
Mix well prior to taking samples of solution for testing.
Do this for 10 min., 20 min., 30 min., and 40 min. after the addition of 1 ml. starch solution.
Observe and record color of solutions in the table provided below.


| C o n c e n t r a t i o n s
Time | 1:10 1:100 1:1000 1:10000









QUESTIONS


1. Describe the predicted/expected results for the investigation of the effect of temperature on enzyme activity. Did you obtain predicted/expected results? If not, what are the predicted/expected results and can you make any reasonable suggestions why you did not get them? (10)

















2. Produce a graph showing time (x-axis) vs. results of Iodine Tests (y-axis) for each of the three temperatures (three labelled temperature plots on one graph). Use the numbers quantifying the color reactions on p. 2. Attach graph to this lab report.

Produce a similar graph showing time (x-axis) vs. results of the Benedicts Tests(y-axis) for each of the three temperatures (three labelled temperature plots on one graph). Use the numbers quantifying the color reactions on p. 2. Attach graph to this lab report (20 pts.)


3. Describe the predicted/expected results for the investigation of the effect of enzyme concentration on enzyme activity. Did you obtain predicted/expected results? If not, what are the predicted/expected results and can you make any reasonable suggestions why you did not get them? (10)