Experiment: To examine the effect of pH on the digestion of protein by pepsin.
Apparatus:
•
Hard-boiled egg-white cut into ¼ inch (or 1 cm) cubes or liquid egg-white suspension?
•
Test-tubes
•
Water-bath at 37oC (or beaker, tripod, burner and thermometer)
•
Stop-watch / stop-clock
•
Buffer solutions (at pH 2, 3, 4, 5, 6, 7 and 8) or: 1M sodium carbonate solution and 2M
HCl (also try 2M NaOH?).
•
1 % (?) pepsin solution (25 ml per pair, minimum 10 ml)
•
Universal indicator paper and colour chart
METHOD
1.
Half-fill one tube with water and another with 50:50 water : pepsin solution.
2.
Quarter-fill a series of labelled tubes with each of the buffer solutions and then fill to
half full with pepsin solution.
3.
Place a piece of egg white or 5 ml of egg white suspension in each tube and place
the tubes in a water bath at 37oC.
4.
After 5 minutes compare the appearance of the contents of each tube.
5.
Graph time to complete hydrolysis against pH and decide which is the optimum pH
for this enzyme.
Wear eye protection when handling acidic and alkali buffers!
Strong alkali buffers are corrosive!
Irritant – acidic buffers!
Experiment:: To examine the effect of pH on amylase action.
Apparatus:
1.
Buffer solutions at pH 10, 9, 8, 7, 6, 5, 4, 3, 2 (or at least 9, 8, 7, 6,5) or: dil HCl, dil
NaOH, dil sodium carbonate and dil citric acid / ethanoic acid
2.
Universal indicator paper and colour chart
3.
1% starch solution (100 ml per pair)
4.
Amylase solution (25 ml per pair)
5.
Boiling tubes + rack
6.
Test tubes
7.
Stop clock
8.
Iodine solution (I2 in KI)
9.
Spotting tile (or watch glass on white surface)
10. Beaker for water bath
11. Water bath at 370C (or tripod, heat mat, thermometer and burner)
METHOD:
1.
Half-fill the test-tubes with each buffer solution.
2.
Add an equal volume of starch solution. Shake well.
3.
Add a few drops of amylase to each tube. Shake.
4.
Test samples from each tube every two minutes by placing a few drops in a watch
glass (or on a spotting tile) and adding iodine solution.
5.
Graph time to complete hydrolysis against pH and decide which is the optimum pH
for this enzyme.
Wear eye protection when handling acidic and alkali buffers!
Strong alkali buffers are corrosive!
Irritant – acidic buffers!
Experiment: To examine the effect of pH on the digestion of protein by pepsin.
Apparatus:
1.
Hard-boiled egg-white cut into ¼ inch (or 1 cm) cubes or liquid egg-white suspension?
2.
Test-tubes
3.
Water-bath at 37oC (or beaker, tripod, burner and thermometer)
4.
Stop-watch / stop-clock
5.
Buffer solutions (at pH 2, 3, 4, 5, 6, 7 and 8) or: 1M sodium carbonate solution and 2M
HCl (also try 2M NaOH?).
6.
1 % (?) pepsin solution (25 ml per pair, minimum 10 ml)
7.
Universal indicator paper and colour chart
Method using buffer solution:
•
Half-fill one tube with water and another with 50:50 water : pepsin solution.
•
Quarter-fill a series of labelled tubes with each of the buffer solutions and then fill to
half full with pepsin solution.
•
Place a piece of egg white in each tube and place the tubes in a water bath at 37 oC.
•
Record the times at which the egg-whites are dissolved.
Method using acid and alkali solutions:
1.
In each of five test-tubes place 5 ml of egg white suspension.
2.
Using a small syringe add acid or alkali as indicated below:
Tube 1. 2.0 ml of sodium carbonate solution
Tube 2. 0.5 ml of sodium carbonate solution
Tube 3. No acid or alkali
Tube 4. 1.0 ml hydrochloric acid
Tube 5. 1.0 ml hydrochloric acid
3.
Using another syringe add 1 ml of pepsin solution
4.
Place all 5 tubes in the water bath at 37oC.
5.
Compare the pH of each tube using universal indicator paper and a colour chart.
6.
After 5 minutes compare the appearance of the contents of each tube.
7.
Graph time to complete hydrolysis against pH and decide which is the optimum pH for
this enzyme.
Experiment:: To examine the effect of pH on amylase action.
Apparatus:
1.
Buffer solutions at pH 10, 9, 8, 7, 6, 5, 4, 3, 2 (or at least 9, 8, 7, 6,5) or: dil HCl, dil
NaOH, dil sodium carbonate and dil citric acid / ethanoic acid
2.
Universal indicator paper and colour chart
3.
1% starch solution (100 ml per pair)
4.
Amylase solution (25 ml per pair)
5.
Boiling tubes + rack
6.
Test tubes
7.
Stop clock
8.
Iodine solution (I2 in KI)
9.
Spotting tile (or watch glass on white surface)
10. Beaker for water bath
11. Water bath at 370C (or tripod, heat mat, thermometer and burner)
Method using buffers:
1.
Half-fill the test-tubes with each buffer solution.
2.
Add an equal volume of starch solution. Shake well.
3.
Add a few drops of amylase to each tube. Shake.
4.
Test samples from each tube every two minutes by placing a few drops in a watch
glass (or on a spotting tile) and adding iodine solution.
5.
Graph time to complete hydrolysis against pH and decide which is the optimum pH
for this enzyme.
Method using acid and alkali solutions: not sure exactly? Presumably similar to pepsin
method?