2.4.3 Enzyme Activity: pH

• 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 (ie. 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 form less easily
  • Eventually, enzyme-substrate complexes can no longer form at all
  • At this point, complete denaturation of the enzyme has occurred

• Where an enzyme functions can be an indicator of its optimal environment:
  • Eg. 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, not surprisingly, is pH 2

The effect of pH on the rate of an enzyme-catalysed reaction for three different enzymes (each with a different optimum pH)

• 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 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

Investigating the effect of pH on enzyme reaction rates

• 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

Iodine can be used qualitatively to indicate the presence or absence of starch from a sample

Apparatus

• Test tubes
• Buffer solutions at different pH levels
• Amylase solution
• Iodine solution
• Starch solution
• Pipettes
• Spotting tile
• Timer
• Gloves
• Goggles

Method

• Wear goggles and gloves
  • Enzymes have the potential to cause allergic reactions if they come into direct contact with 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 volume and concentration of the substrate (starch) should be used so these variables are controlled and the effect of 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 the mixture on the second drop of iodine
• Repeat every 10 seconds until iodine solution remains orange-brown
  • When the solution remains orange-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 the better the enzyme works at that pH

Investigating the effect of pH on enzyme activity

Limitations

• The above method can be adapted to control temperature by using a water bath at 35℃
  • 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