Denaturing of the active site
Enzymes are proteins. Proteins are made up of a series of amino acids forming a polypeptide chain. The order of these amino acids is called the primary structure of the protein. The secondary structure of the protein is the regular coiling or folding of the polypeptide chain forming an alpha helix or a beta pleated sheet. However the important thing to consider when exercising this investigation is the protein’s tertiary structure.
The tertiary structure of a protein results from 3D coiling of the already folded chain of amino acids. It is formed by chemical bonds between the R groups of the amino acids and the protein folds into an irregular 3D shape. Proteins with tertiary structures are known as globular proteins. Since the sequence of amino acids is different in every protein, the bonds form in different places and therefore each globular protein has a different shape. This is why enzymes are specific.
As can be seen in the above diagram, it is the active site of the enzyme where the reaction occurs. It is the shape of the active site that must be specific (due to the enzymes tertiary structure) so that the substrate fits into the active site and the reaction can occur. The specific shape of the active site is due to the bonds between the R groups. However these bonds can be broken by high temperatures and therefore the active site can lose it’s specific shape and become denatured. When the enzyme has become denatured, the substrate will no longer fit into the active site and therefore a reaction is not able to take place. This means as more enzymes denature due to increasing temperature, the rate of reaction will decrease.
Like the body, enzymes have an optimum working temperature. The optimum temperature for an enzyme is about 40 degrees Celsius. Our bodies stay at a temperature of around 37 degrees C which means enzymes in the body will always be working close to their optimum temperature and therefore as quickly as possible. It would be dangerous for the body to have a working temperature of 40 degrees C as if the temperature was raised even slightly, enzymes would start to denature.
Graph to show the effect of temperature on the activity of enzymes
Prediction
Having looked at the activity of enzymes and reactions in detail, I can make a knowledgeable prediction on the outcome of the experiment. I predict that as I increase the temperature of the both the milk and the rennin solution before and during mixing, the rate of reaction will increase. This will be due to there being more kinetic energy as the temperature rises so more collisions between the enzyme and substrate molecules, more enzyme-substrate complexes formed and therefore a quicker reaction. This will be shown in my experiment by the time the rennin solution and milk takes to coagulate.
However I also expect there to be an optimum point in my experiment where the enzyme works at it’s fastest, and then as the temperature rises after that point I will expect the rate of the reaction to decrease due to the active sites of the enzymes becoming denatured so less enzyme-substrate molecules being formed and a slower reaction. I expect this optimum point to be about 40 degrees Celsius, as this is most animal/human enzymes optimum temperature. This will be shown in my experiment if the mixture that is quickest to coagulate is that nearest to 40 degrees Celsius.
Safety
It is imperative that the experiment I carry out is done so in safe conditions. There are measures I can take to ensure that this is the case.
- All coats and bags must be well out of the way of the area being used, as they may cause someone to trip over them causing potential injury.
- All liquid must be handled carefully so as to prevent spillages as this could cause potentially dangerous slippery surfaces. If something is to be spilt, it is to be wiped up immediately.
- Glass beakers/test tubes will be in use so careful handling is vital. If something is to smash, it needs to be cleaned up immediately, yet cautiously.
- There should be no running in the lab at any time.
- As electronic water baths are being used extreme care must be taken, as spillages of water near electrical sockets can be highly hazardous.
- Although the enzyme solution is a low dilution, it may cause an allergic reaction so avoid skin contact and rubbing eyes with contaminated hands.
Equipment
Below is a table showing the required equipment for my experiment and also the reason why I have chosen to use that equipment.
Method
I will test each of the temperatures 3 times to make sure that anomalous results can be identified and an average can be calculated for each temperature. To make my experiment as efficient as possible I will do all the tests of one temperature altogether, before moving on to the next temperature. So I will need to produce a method testing 3 rennin solution and milk mixtures at the same temperature at the same time, which I will repeat 5 times in all (for water at room temperature, 30, 40, 50 and 60 degrees C).
- Place 3 test tubes into a test tube rack.
- Using the pippet, measure 5 cm 1% rennin solution into each test tube.
- Label the test tubes as 30 degrees Celsius with the marker pen.
- Place the test tubes into the 30 degree Celsius electronic water bath.
- Place 3 new test tubes into the test tube rack.
- Using the syringe measure 5 cm milk into each test tube.
- Place the test tubes into the same water bath (30 degrees C).
- Leave the milk and rennin solution to equilibrate for 10 minutes.
- Check and record the temperature of the water in the water bath, by leaving a thermometer standing in the water bath, as it may not be exactly accurate to 30 degrees.
- Using another thermometer, check that the milk and rennin solutions have equilibrated to the temperature of the water bath.
- Mix the rennin solution and the milk by pouring the rennin solution into the milk then pouring the mixture back into the test tube which contained the rennin solution and replace in the water bath. Do this for each test tube as quickly as possible.
- Immediately start the stop clock.
- Check the contents of the tubes every 30 seconds by holding the tube against a piece of black card and tilting gently. When the milk proteins begin to coagulate you will see small granules appearing in the milk.
- Record the time taken for the first signs of coagulation of the milk proteins. This will be to the nearest 30 seconds as it would be difficult to time the reaction to the nearest second.
Repeat this whole method using water baths at 40, 50 and 60 degrees Celsius as well as at room temperature (using no water bath).
The variables in this experiment are the temperature of the water and the time taken for the mixture to coagulate. The temperature of water is the independent variable as it was decided which temperatures would be used before carrying out the experiment. The time taken for the mixture to coagulate is the dependant variable as the outcome of this depends on the temperature of the water and the results are not known prior to the experiment.
Fair test
For this to be a successful and reliable investigation it is imperative that I take care and consider the following factors to ensure that it will be a fair test.
- The same equipment must be used throughout the whole experiment. This includes the same water baths, syringe, pippet, thermometers, stop clock and black card. This ensures that if an error occurs due to a discrepancy of a piece of equipment, the same error will occur on all tests.
- The temperature of the water baths must be that of what is stated in the method. Measured amounts must also be precise, as discrepency in measurements could lead to anomolous data.
- All timings must stay consistent and exact throughout the investigation to make sure all the results are to the nearest 30 seconds.
To ensure this is a fair test there must only be one variable factor. In this investigation the variable factor will be the different temperatures used. I will keep all the other factors the same throughout. The concentration of rennuin solution will remain constant throughout (at 1%) as I will take all the measured samples of rennin solution from the same beaker of solution.
When I have completed my experiment, I will use my obtained results to calculate an average time of coagulation for each temperature. I will then use these averages to plot a graph to show the effect of temperature on the activity of the enzyme rennin. I will then be able to assess the accuracy of my prediction and the success of my experiment as well as come to a conclusion of the initial question.
Bibliography
Biology 1 – Jones, Fosbery, Taylor