Enzyme Rate Practical (AQA AS Biology): Revision Note
Exam code: 7401
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
Place single drops of iodine solution in the dips on a spotting tile
Label a test tube with the pH to be tested
Use a syringe to place 2 cm3 of amylase in the test tube
Add 1 cm3 of buffer solution to the test tube using a syringe
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
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
Wipe the glass rod with paper towel and wait 10 seconds before placing another drop of the mixture on the second drop of iodine
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
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
Set up two test tubes and label to indicate that one contains bile salts and the other does not
Add 5 drops of phenolphthalein to each tube
Phenolphthalein is an indicator that turns pink alkaline and colourless in an acid
Add 5 cm3 of milk to each tube
Milk contains lipids, so functions as a lipid 'solution'
Add 7 cm3 of sodium carbonate solution to each test tube
The resulting solution should be pink, as sodium carbonate is alkaline
Add 1 cm3 of lipase to the first test tube and start the stopwatch
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
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
Examiner Tips and Tricks
When describing control variables, remember to always refer to the volume and concentration of a solution; you should never use the word 'amount' in this context.
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