3.6: Enzyme Kinetics (Activity)

The Enzyme

Amylase is an enzyme that breaks down amylose (starch) into glucose molecules.

1. What test can be used to indicate the presence of Starch?
2. What parameters would influence the ability of the enzyme to facilitate the rate of the reaction?
3. What is the role of an enzyme in a chemical reaction and what is it made of?
4. What test can be used to indicate the presence of glucose?

Salivary amylase is produced in the mouth, where digestion begins.

Pancreatic amylase is produced in the pancreas and is supplied to the duodenum of the small intestines.

Overlay of salivary (green) and pancreatic (teal) amylase molecules.

Effect of Temperature on Enzyme Activity

1. Add 5 ml of H₂O to a tube (this is the BLANK).
2. Add 5 ml of starch (substrate) to 3 separate tubes.
   • One on ice (0°C), one on the bench (25°C), and one in a 40°C water bath
3. Add 2 drops of iodine to each tube and mix: Blank, 0°C, 25°C, and 40°C.
4. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm.
5. Read each tube in the spectrophotometer. This is time 0 minute.
6. Add 35 drops of amylase solution to each tube simultaneously. Mix and ensure incubation is occurring at the correct temperature.
7. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer and immediately return the tubes to the proper temperature.
8. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time (min)	0ºC % Trans	25ºC % Trans	40ºC % Trans
0
2
4
6
8
10
12
14
16
18
20
22

Effect of pH on Enzyme Activity

1. Add 5 ml of water to an empty tube (this is the BLANK)
2. To 3 separate tubes, add 2.5 ml of buffer pH 3, pH 5 or pH 7
   1. Add 2.5 ml of starch (substrate) to each of these tubes (excluding BLANK)
3. Add 2 drops of iodine to each tube and mix: Blank, pH 3, pH 5, pH 7
4. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm
5. Read each tube in the spectrophotometer. This is time 0 min.
6. Add 35 drops of amylase solution to each tube simultaneously, mix to homogeneity.
7. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer.
8. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time (min)	pH 3 % Trans	pH 5 % Trans	pH 7 % Trans
0
2
4
6
8
10
12
14
16
18
20
22

Effect of Enzyme Concentration on Enzyme Activity

1. Add 5 ml of water to an empty tube (this is the BLANK)
2. Add 5 ml of pH 7 buffer to 3 separate tubes.
3. Follow the dilution scheme below:

Dilution scheme for amylase (enzyme)

4. In 4 separate tubes, ADD 4 ml of starch solution.
   • Label them 1x, 1/5x, 1/25x, 1/125x
5. Add 2 drops of iodine to each starch tube and the Blank.
6. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm.
7. Read each tube in the spectrophotometer. This is time 0 minute.
8. Add 35 drops of diluted amylase solutions to the appropriately labeled tubes simultaneously and mix.
   • Each tube receives a different Amylase dilution.
9. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer.
10. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time (min)	1 X % Trans	1/5 X % Trans	1/25 X % Trans	1/125 X % Trans
0
2
4
6
8
10
12
14
16
18
20
22

Effect of Substrate Concentration on Enzyme Activity

1. Add 5 ml of water to an empty tube (this is the BLANK)
2. Add 5 ml of pH 7 buffer to 3 separate tubes
3. Follow the dilution scheme below:

4. In 4 cuvettes, ADD 4 ml of the diluted starch solution.
   • Label them 1x, 1/2x, 1/4x, 1/8x.
5. Add 2 drops of iodine to each starch tube and the Blank.
6. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm.
7. Read each tube in the spectrophotometer. This is time 0 minute.
8. Add 35 drops of amylase solution to each tube simultaneously and mix.
9. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer.
10. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time (min)	1 X % Trans	1/2 X % Trans	1/4 X % Trans	1/8 X % Trans
0
2
4
6
8
10
12
14
16
18
20
22

Plot the Results

1. Using a plot.ly, plot the data on the same chart.
2. Calculate the line of best fit of each dataset.
3. The slope represents the activity of the enzyme in each condition. What is the unit of this activity?