OCR AS Biology

Revision Notes

2.4.3 Enzyme Activity: pH

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 an enzyme-catalysed reaction, downloadable AS & A Level Biology revision notes

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

Using iodine to test for starch, IGCSE & GCSE Biology revision notes

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 effect pH on enzyme activity, IGCSE & GCSE Chemistry revision notesInvestigating 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

Exam Tip

Temperature can both affect the speed at which molecules are moving (and therefore the number of collisions between enzyme and substrate in a given time) and can denature enzymes (at high temperatures). pH, however, does not affect collision rate but only disrupts the ability of the substrate to bind with the enzyme, reducing the number of successful collisions until eventually, the active site changes shape so much that no more successful collisions can occur.

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