2.4.3 Enzyme Activity: PH

Question 1

Denaturing due to pH

Answer 1

All enzymes have an optimum pH or a pH at which they operate best
Enzymes are denatured at extremes of pH
- hydrogen and ionic bonds hold the tertiary structure of the protein (i.e. the enzyme) together
- below and above the optimum pH of an enzyme, solutions with an excess of H+ ions (acidic solutions) and OH- ions (alkaline solutions) can cause these bonds to break
- the breaking of bonds alters the shape of the active site, which means enzyme-substrate complexes from less easily
- eventually, enzyme-substrate complexes can no longer form at all
- at this point, complete denaturing of the enzyme has occurred

Question 2

Optimum pH

Answer 2

Where an enzyme functions can be an indicator of its optimal environment:
- e.g. pepsin is found in the stomach, an acidic environment at pH 2 (due to the presence of hydrochloric acid in the stomach’s gastric juice)
- pepsin’s optimum pH, is therefore pH 2

Question 3

Buffer Solutions

Answer 3

When investigating the effect of pH on the rate of an enzyme-catalysed reaction, you can use buffer solutions to measure the rate of reaction at different pH values:
- buffer solutions each have a specific pH
- buffer solutions maintain this specific pH, even if the reaction taking place would otherwise cause the pH of the reaction mixture to change
- a measured volume of the the buffer solution is added to the reaction mixture
- this same volume (of each buffer solution being used) should be added for each pH value that is being investigated

Question 4

Investigating the Effect of pH on Enzyme Reaction Rates

Answer 4

Use the enzyme amylase to breakdown starch at a range of pH values, using iodine solution as an indicator for the reaction occurring
- amylase is an enzyme that digests starch (a polysaccharide of glucose) into maltose (a disaccharide of glucose)
A continuous sampling technique can monitor the progress of the reaction
- starch can be tested for using iodine solution

Question 5

PH Investigation Apparatus

Answer 5

• test tubes
• buffer solutions at different pH levels
• amylase solution
• iodine solution
• starch solution
• pipettes
• spotting tile
• timer
• gloves
• goggles

Question 6

PH Investigation Method

Answer 6

Wear goggles and gloves
- enzymes have the potential to cause allergic reactions if they come into direct contact with the skin
Place single drops of iodine solution in rows on the tile
- iodine solution is orange-brown
Label a test tube with the pH to be tested
Use the syringe to place 2cm3 of amylase in the test tube
- equal volume and concentration of enzyme should be used so these variables are controlled and the effect of changing pH can be measured
Add 1cm3 of buffer solution to the test tube using a syringe
Use another test tube to add 2cm3 of starch solution to the amylase and buffer solution, start the stopwatch whilst mixing using a pipette
- equal concentration and volume of the substrate (starch) should be used so these variables are controlled and the effect changing pH can be measured
- mixing enables the enzymes and substrate to be equally mixed
After 10 seconds, use a pipette to place one drop of the mixture on the first drop of iodine, which should turn blue-black
- this test indicates whether starch is still present
Wait another 10 seconds and place another drop of type mixture on the second drop of iodine
Repeat every 10 seconds until iodine solution remains organs-brown
- when the solution remains organs-brown, it means amylase has broken down all of the starch so nothing is left to react with the iodine
Repeat experiment at different pH values
- the less time the iodine solution takes to remain orange-brown, the quicker all the starch has been digested and so better the enzyme works at that pH

Question 7

Limitations of Enzyme pH Investigation

Answer 7

The method can be adapted to control temperature by using a water bath at 35 degrees C
- all solutions that need to be used (starch, amylase, pH buffers) should be placed in a water bath and allowed to reach the temperature (using a thermometer to check) before being used
A colorimeter can be used to measure the progress of the reaction more accurately
- with a solution containing starch being darker and glucose lighter (as a result of the colour-change of iodine)
- this will affect the absorbance or transmission of light in a colorimeter