3-3-3-enzyme-rate-practical

Factors affecting digestive enzymes

• It is possible to investigate the effect of relevant factors on the rate of digestive enzyme activity, e.g.

  • pH

  • the presence of bile salts

Investigating the effect of pH on amylase activity

• Amylase digests starch into maltose, meaning that the iodine test can be used to measure the progress of the reaction

  • Strong positive iodine test = starch is present at high concentrations, so amylase activity is absent, or very low

  • Weak positive iodine test = starch is present at a low concentration, so amylase activity is high

  • Negative iodine test = no starch remaining, so amylase has been active and all starch has been converted to maltose

Apparatus

• Spotting tile

• Iodine solution

• Test tubes

• Starch solution

• Buffer solutions at a range of pH levels

• Amylase solution

• Glass rod

• Timer

• Paper towel

• Gloves

• Goggles

Method

1. Place single drops of iodine solution in the dips on a spotting tile

2. Label a test tube with the pH to be tested

3. Use a syringe to place 2 cm³ of amylase in the test tube

4. Add 1 cm³ of buffer solution to the test tube using a syringe

5. Add 2 cm³ of starch solution to the amylase and buffer solution; immediately start the stopwatch whilst mixing the tube contents with a glass rod

   • Mixing ensures that the enzymes and substrate are evenly distributed

6. After 10 seconds, use a glass rod to place one drop of the mixture on the first drop of iodine, in the tile

   • This should turn blue-black, indicating that starch is still present

7. Wipe the glass rod with paper towel and wait 10 seconds before placing another drop of the mixture on the second drop of iodine

8. Repeat step 7 every 10 seconds until the iodine solution remains orange-brown

   • This indicates that the amylase has broken down all of the starch

9. Repeat steps 1-8 at different pH values; the less time the iodine solution takes to remain orange-brown, the quicker all the starch has been digested

   • Ensure that control variables remain constant for each repeat, e.g.:

     • Equal volume and concentration of enzyme solution

     • Equal volume and concentration of the substrate solution

     • Equal volumes of buffer solution

     • The same stirring method

     • Cleaning of the glass rod should be thorough

Limitations

• Determining that point at which the iodine no longer changes colour can be subjective, so a colorimeter can be used to measure the progress of the reaction in an objective way

Investigating the effect of bile salts on lipase activity

• Bile salts aid the process of emulsification in lipid digestion

• Experiments can be conducted to investigate the effect of bile salts on the rate of lipase activity

Apparatus

• Measuring cylinders or syringes

• Beakers of volume 100 cm³

• Test tubes

• Glass rod

• Stopwatch

• milk, full-fat or semi-skimmed, 5 cm³

• Phenolphthalein indicator in a dropper bottle

• 5 % lipase solution

• Sodium carbonate solution, 0.05 mol dm^–3

• Bile salts, or washing up liquid (which also causes emulsification)

Method

1. Set up two test tubes and label to indicate that one contains bile salts and the other does not

2. Add 5 drops of phenolphthalein to each tube

   • Phenolphthalein is an indicator that turns pink alkaline and colourless in an acid

3. Add 5 cm³ of milk to each tube

   • Milk contains lipids, so functions as a lipid ‘solution’

4. Add 7 cm³ of sodium carbonate solution to each test tube

   • The resulting solution should be pink, as sodium carbonate is alkaline

5. Add 1 cm³ of lipase to the first test tube and start the stopwatch

6. Stir continuously while observing the colour of the test tube contents; record the time at which the solution is no longer pink, but has become white

   • As lipase breaks down the lipids into fatty acids and glycerol, the pH of the milk decreases until it becomes slightly acidic

   • When phenolphthalein becomes colourless, the white colour of the milk becomes visible again

7. Repeat steps 1-6, but this time add 1 or 2 drops of bile salts, or washing up liquid, to the test tube and stir before adding lipase

   • Ensure that control variables remain constant for each repeat, e.g.:

     • Equal volume and concentration of enzyme solution

     • Equal volume of milk

     • Milk of the same fat content

     • Equal volume and concentration of sodium carbonate solution

     • Equal volume of phenolphthalein

     • The same stirring method

     • The same person deciding the point at which the milk has become colourless / the same white coloured paper used as a comparison point