enzyme practicals Flashcards
(20 cards)
what can we test the effect of substrate concentration on
catalase activity
method for effect of substrate concentration on catalase activity
1) make up dilutions of hydrogen peroxide in the boiling tubes using table 1. there should be 6 test tubes with 10cm3 per tube
2) add 1cm3 of celery extract into a watch glass and soak 3 filter papers in the extract for 5 minutes
3) remove one paper disc using the forceps, shake off excess enzyme and drop it into the first hydrogen peroxide tube till it reaches the bottom
4) start the stopwatch when it hits the bottom and record the time it takes to reach the surface
5) repeat with the other two discs
6) discard the celery extracts with a new one and repeat steps 2-5 for the next dilutions
what caused the paper discs to lift from the bottom of the tube
the hydrogen perioxide reacted with catalase enzyme in the celery extract to produce water and oxygen. the oxygen formed pushed the paper disc upwards causing it to rise to the surface
explain the results obtained for the different substrate concentrations
as the concentration of substrate/hydrogen peroxide increased, the time taken for the discs to rise to the surface decreased. therefore the rate of reaction increased. the increase in substrate concentration meant more substrate molecules were able to successfully collide with the catalase enzyme (celery extract) to produce more enzyme-substrate complexes and ultimately form more product. one of the products was oxygen so the increase in product/oxygen formation meant that more oxygen could push the paper disc further so the disc took a shorter amount of time to rise to the surface therefore increasing the rate of reaction
give limitations of the procedure and how you would overcome them (1) (substrate conc)
- one limitation of the procedure was that when we repeated the experiment with the two other discs we used the same boiling tube containing the hydrogen peroxide from the previous trial with the other discs. this meant that there was less substrate every time we repeated the experiment with the other two discs and it meant it would take slightly longer for the disc to rise because less substrate means less successful collisions can occur.
we could use a completely new dilution for each paper disc that we trial to avoid the random error of putting each trial disc in the same boiling tube of hydrogen peroxide dilution
give limitations of the procedure and how you would overcome them (2) (substrate conc)
- another limitation was when all 3 discs were placed in the celery extract, we had to trial each disc one at a time so some discs were left in the celery extract for a longer time so the discs used later would have had a slightly higher enzyme concentration which could have caused the paper discs to rise to the surface quicker than the other two
after leaving the enzyme discs in the celery extract for 5 minutes we could take them all out at the same time to avoid the other two discs being left in the extract for a longer time
which limitation in the substrate conc practical cant be overcome
we used celery extract as the catalase enzyme. this means its not 100% catalase so could mean the number of successful collisions would decrease than what it should have been regardless of how much you increase the substrate concentration
define systematic errors and suggest which of your errors fall into this category
they refer to an error in measurement that either remains constant or changes in a predictable manner with changing conditions. they are consistent repeatable errors involving equipment. in this case, the systematic error would be thatr the paper disc wouldnt go directly to the bottom so we werent accurately measuring the time it took for the disc to rise from the bottom of the boiling tube to the top
how to work out rate of reaction
1/time
method for investigating the effect of pH on catalase activity
1) set up 5 inverted 100cm3 measuring cylinders by filling with water and inverting it into a large beaker.
2) insert the delivery tube into the open end of the measuring cylinder under water
3) label 5 tubes with the pH buffers 4,5,6,7 and 8 and add 1cm3 of pH buffer to the appropiately labelled tube using a clean 1cm3 syringe each time
4) now add 10cm3 of hydrogen peroxide to each tube
5) using a clean 10cm3 syringe quickly add 1cm3 of the enzyme catalase to each tube and inser the rubber bung and the side arm delivery tube. start the stopwatch - it is important to do these steps as quickly as possible
6) the other end of the delivery tube should be completely under the inverted measuring cylinder
7) time the gas given off for 15 minutes and then read the volume of gas collected for each tube
explain why changes in pH alter the rate of an enzyme controlled reaction
enzymes have an optimal pH at which they work best. if the pH changes, it affects the enzymes since the hydrogen ions in an acidic solution or hydroxide ions in an alkaline solution disrupt the tertiary structure of the active site. this is because hydrogen and ionic bonds are broken. the active site changes shape and denatures reducing the number of enzyme substrate complexes forming and can reduce the rate of reaction
explain why different enzymes are affected by pH changes in different ways
different enzymes have different optimal pH values. enzymes in the stomach work best in acidic conditions and enzymes in the small intestine work best in alkaline conditions. changes in pH can alter changes in enzyme structure. if enzymes with a neutral optimal pH are surrounded by an acidic solution the high proportion of H+ ions will disrupt the ionic bonding making the enzyme lose its shape and so it denatures becoming inactive
main limitations of the procedure and how they affect the data (pH)
- gas leaks in the setup so water may have leaked from the measuring cylinder (we’ve underestimated the amount of oxygen produced)
- delays in adding the enzyme and starting the stopwatch could cause error as it could be that gas was collected before starting the timer
- not controlling temperature could lead to changes in reaction. they may have incorrectly read of the measuring cylinder
how to overcome the limitations (pH)
use a gas syringe instead of a measuring cylinder
ensure that is the measuring cylinder is tightly shut to prevent gas escaping
maintain a constant temperature using a water bath
repeat the experiment a few times
method for investigating the effect of temperature on amylase activity
1) prepare water baths
2) add 10cm3 of starch to each boiling tube labelled 0°C, 20 °C, 40 °C, 60 °C, 80 °C. add each tube to each corresponding water bath
3) add 1cm3 of amylase to each test tube (labelled with the same temperatures)
4) leave all the test tubes for 5 minutes
5) add two drops of iodine to the wells of the spotting tile
6) after 5 minutes, add the enzyme to the starch and stir with as glass rod. start the stopwatch
7) take two drops of the enzyme-substrate sample and add it to the first tile
8) take samples every minute for 10 minutes
9) leave 5 minutes before mixing the starch and amylase for the next temperatures
10) repeat steps 6-8 for all temperatures
explain why the starch was made up with a buffer solution (temp)
the buffer solution maintains a constant pH so ensures that there are no changes in acidity or alkalinity that could also affect the rate of reaction
why were all the boling tubes and test tubes left in the water bath five minutes before mixing the enzyme with the starch (temp)
it allows both the starch and amylase solutions to reach a desired temperature before the reaction begins to ensure high validity of the results
describe and explain the results obtained for the different temperatures
as temperature increases, kinetic energy increases which increases collisions between the substrate and the enzyme. more successful collisions leads to an increase in the number of ESC forming and increases the amount of product formed. as temperature is above the optimal, the bonding between the enzyme and the substrate is damaged and the active site denatures. the active site is no longer complementary to the substrate so the number of enzyme-substrate complexes that form decreases which decreases the amount of product formed
limitations of the temp practical
- uneven mixing of enzyme and starch solution which can lead to differences in enzyme concentration
- difficult to control temperature in water bath
- cant determine the exact time when the starch has fully broken down
- possible human error in timing and interpreting the colour of the iodine test
how to overcome limitations of the temp practical
- use an electronic water bath with precise temperature control
- use a colorimeter to measure colour changes objectively
