The Effect of Temperature on Enzyme Activity
Aim: To investigate how temperature effects the enzyme catalase.
Hypothesis: If the temperature of water is increased then the enzyme will react quicker to form oxygen and water, when compared to cold water.
Purpose: To design and conduct a plan of a practical about the effects of temperature on enzymatic activity with a partner.
Introduction: An enzyme is a protein, which speeds up a specific chemical reaction without altering the products of the reaction and without being altered itself (LeCornu and Diercks, 2011). Enzymes catalyse a reaction by lowering the activation energy (LeCornu and Diercks, 2011). A number of factors may change an enzyme’s environment such as temperature, pH, surface area and the presence of competitive inhibitors and non-competitive inhibitors. (LeCornu and Diercks, 2011). Change to an enzyme’s shape alters the activation site so that it is no longer complementary to its substrate and denatures the enzyme. This experiment will focus on the effect of temperature on the activity of catalase.
Hydrogen peroxide is a toxic by-product found in cells. Catalyse is an enzyme breaks down hydrogen peroxide into oxygen and water in the liver. When this reaction occurs oxygen gas is given off and creates foam (Buddies, Science, 2016). The balanced equation for the reaction is shown below. 2H2O2 —>2H2O + O2
The volume of oxygen produced is directly proportional to the rate catalase breakdowns hydrogen peroxide. In this investigation this will be measured by the height of the foam produced in each measuring cylinder.
The optimum temperature of Catalyse is 37 degrees Celsius, as this is the temperature it functions in the body. Therefore it can be inferred that enzyme will work the quickest and produce the most foam in 27-37 °C and the higher or lower temperatures will decrease the rate of the reaction of Catalyse.
Independent variable: Temperature of water
Dependent variable: The rate catalase activity. This can be determined by the height of the foam (mm) produced in from the reaction. As the foam is an indication of the oxygen produced from the breakdown of hydrogen peroxide in the reaction. Therefore measuring the foam is a measure of the rate catalytic activity of catalase.
Constant Variables: •Volume of water •Volume of hydrogen peroxide •Temperature of water baths for each trial
Materials: •4x 250ml glass beakers •3x 100mL measuring cylinders •81ml hydrogen peroxide pH 7 •1x plastic dropper •1x 10ml small glass measuring tube •2x drops of detergent from a dropper bottle per measuring cylinder •1x1x1cm cubes of liver •Scalpel (to cut liver cubes) •1x ceramic tile •3x tweezers (to pick up and transfer liver from place to place) •Pair of latex gloves, safety glasses and apron per person •Stop watch or phone – to time and take photographic evidence of steps taken in practical •1x ruler (to measure sides of liver and for in case when amount of foam exceeds measurement marks on measuring cylinders) •Paper towels (as many as you need to wrap liver and wipe down work space) •1x bottle of Dettol (to pour on paper towels to wipe down work space after practical) •Paper and pen to record results
•OPTIONAL – pin board to pin up paper for results and method •2x thermometer Method:
1.Prepare liver on tile and use a scalpel to cut nine cubes that are 1cm-1cm-1cm, use a ruler to measure the sides accurately.
2.Gather materials and rinse the glassware with distilled water and dry with a paper towel.
3.Pour up to 250ml of cold water at 27 °C in each of the three beakers. If the water is too cold, set it aside for a short time so the temperature will drop to 27 °C, repeatedly check the thermometer in the water (it must be left in there for at least 10 sec) to make sure it is the right temperature. If the water is too warm add more cold water. There should only be 250ml water in each beaker so pour out excess water if any.
4.Label measuring cylinders as 1, 2 and 3. Set out beakers next to each other in a straight row and record the temperature of the water in the results table. Check temperatures to ensure they are all the same using the thermometer. Place each measuring cylinder in each water-filled beaker.
5.Pour 81 ml of hydrogen peroxide into an empty beaker and label it ‘Hydrogen Peroxide’ to clarify its contents. Get a dripper bottle of detergent and transfer 9 ml of hydrogen peroxide from the beaker using a dropper and place the amount in a small measuring tube. Put two equal drops (as close to equal as possible) of detergent along with the 9ml of hydrogen peroxide in the small measuring tube in each measuring cylinder.
6.Set the time duration for the specific temperature trial on the timer but do not start it yet. Be extremely observant and alert. One person holds x2 tweezers, one in each hand to hold a liver cube and the other person holds a liver cube using x1 tweezers in just one hand whilst they are ready to set the timer with their other hand. Both people hold the liver cubes at the exact same level above the opening of the measuring cylinders and drop the liver cubes into the cylinders at exact same time. Drop the liver cubes on a count of three. Start the timer as soon as the liver touches the surface of the solution.
7.Both observe the reaction for the set time duration and record the total volume of foam, observations and reaction rate (ml/min) in the results table when the time is up.
8.Pour the water out the beakers into the sink and wash out the solution in the large cylinders under running water – this glassware will be reused for next trials. Wrap the used liver in paper towels to be disposed of as biological waste.
9.Repeat this process using warm water at 37 C° then hot water at around 47 °C. The next three unused liver cubes are used for each trial.
10.Once the practical is complete, remove and dispose of the gloves in a bin, rinse the unclean equipment under running water and dry them off then return them to their respective places. Also dispose of the biological waste in a biological waste bin/bag, should already be provided the by the lab supervisor. Lastly, wipe down the work area using paper towels partially soaked in Dettol.
Table 1: The Rate of enzyme activity affected by liver
Height of froth after 1 minute
Graph 1: The effect of temperature on the Rate of enzyme activity
Random Errors A potential source of random error was that the same glassware and equipment was used in each trial and may have not been rinsed properly. This may have contaminated the solution, which would result an increase or decrease of catalase activity. A parallax error may have occurred when measuring the amount of foam produced from each reaction, which decreased the precision of the results. Due to reaction time when using a timer the exact rate of reaction of the catalase activity cannot be determined. The time the liver reacts with the solution varied with the sample of 3 for each temperature. Because the reaction initiates as soon as the reactants make contact catalase activity would be higher than the true value in the results. Another possible error is that the room temperature changes therefore the temperature of the water cannot be kept constant with each trail and produces unreliable results.
Systematic Errors Additionally there is the possibility for systematic errors to occur as the result of incorrect calibration by the manufacturer. For example if the thermometer was calibrated incorrectly, inaccurate measurements, which are higher or lower than the true value, would be produced. By using different apparatus and fresh liver could have highlighted systematic error. Additionally the stopper bottle used cannot accurately measure the same amount of detergent. More detergent would therefore cause more bubbles to form, which produces an inaccurate measurement. The procedure to cut up the liver aimed to measure 1cm- 1cm- 1cm was to improve precision, but this factor could not be made consistent. When there are different sizes of liver, liver with a bigger surface area requires more hydrogen peroxide to break down. In addition the poor accuracy in measuring solutions affects the precision of results.
Sample size and repetition This investigation was conducted with a sample size of 3, to decrease random errors and evaluate the reliability of the experiment. By repeating the experiment and using more sample sizes we were able to formulate an average and remove outliers, which increased the accuracy of the results to the true value. Repeated the experiment several times identifies the presence of any systematic errors and gives information on how close the results is the true value.
Improvements •Electronic machine to keep temperate constant in beaker •Clamp to hold thermometer to prevent from sitting on glass and getting temperature of liver. •Repeat using other animal cells or liver •Machine to precisely cut liver at the same size •Measure volume of the drops of detergent exactly •Measuring room temperature every 10 minutes
Extension to Practical Ways in which to extend the practical and increase knowledge and understanding could include using more sample sizes, looking at other variables which effect liver function for a wider understanding and repeat using other animal cells or liver.
Conclusion: •The hypothesis (is/ is not) supported as (Summarize findings).
Pre planning Throughout the pretrial my partner and I communicated well but discussion and writing note together of our observations. We also had good teamwork by not blaming each other when mistakes were made and used problem-solving skills to find a solution. Next time I think we can manage our time better by delegating roles, such as who sets up glassware and who measures hydrogen peroxide. My partner and I worked best in the planning stage. Xuan contributed to the group in a really positive way by suggesting useful ideas and improvements when designing our practical. We were able to work well together by considering each other’s idea and making joint decisions. Xuan and I were able to delegate tasks for homework. We also communicated and shared updated work via email to stay up to date.
On the day We managed our time well by