Aim: The aim of the experiment is to find out what effect temperature has on the action of a protease enzyme on exposed developed film.
Enzymes are biological catalysts. They are made in livings things built up by amino acids to make protein. Enzymes are able to speed up reactions and can repeat reactions.
There are various factors that affect the activity of enzymes they are:
“Y Concentration of enzyme or substrate
Enzymes are specific, this means that they only work on one substrate molecule. A substrate molecule is what the enzyme actually works on.
The factors I have chosen to investigate are temperature. This therefore means that the temperature will be the independent variable.
In the experiment there will be a transparent plastic backing of developed film, which will have a black gelatine coat on it. The gelatine coat is protein, which is the substrate molecule. I will put the film into protease solution, which is the enzyme. By having the gelatine coat I am able to see what happens to the gelatine coat when the temperature increases. I can find out if temperature affects the action of a protease enzyme.
Enzymes have an optimum temperature, which is generally below 400C. The optimum temperature is when enzymes works best and fastest at. When the temperature rises the rate increases. This is because the substrate and enzyme molecules are moving faster because the temperature has increased. This means that the molecules have more energy. They therefore are likely to collide more often with each other and a reaction will take place. However if the temperature goes over the optimum temperature the reaction slows down and the enzyme denatures. This means that it has changed shape and therefore the substrate can no longer fit into the enzyme.
The diagram below shows how the substrate molecules which is protein fits into the enzyme, which is a protease molecule. This type of mechanism is called the lock and key hypothesis.
If the active site, which is the enzyme, is heated too much it will change shape and no longer fit the substrate. The substrate therefore no longer is able to react if there is no active enzyme.
I predict that when the temperature increases the time taken for the gelatine to be broken down will decrease. This is because temperature is a catalyst, which helps to speed up the enzymes, which are biological catalysts. When the temperature is 300C I predict that it will take longer for the film to become transparent than when the film is in a temperature of 600C. However at a certain temperature in the experiment I predict that there will be an optimum temperature. This is when the enzyme works best at. After this point the enzymes start to slow down and eventually denature which means it is harder for the substrate molecules to fit into the enzyme molecules.
As I predict that when the temperature increases the time taken for the gelatine to be broken down decreases until it reaches the optimum temperature I therefore predict that the rate of reaction will increase when the temperature increases until it reaches the point when the enzymes start to denature.
When the temperature is increased the enzyme molecules will break down the black gelatine coat quicker and therefore the developed film will become transparent faster. When temperature is increased the substrate molecules of protein will collide more frequently with the enzyme molecules. So if the temperature is increased from 300C to 600C the enzyme molecule will break the black gelatine down faster to leave the transparent plastic backing.
The two diagrams show the effect of temperature between substrate molecules and enzyme molecules. They are only rough diagrams of what will happen between the two molecules.
“Y Substrate molecule-
“Y Enzyme molecule-
Apparatus: The apparatus that I am going to use for the experiment will be a test tube, developed film with a gelatine coat, splint, syringe, stopwatch, thermometer and electric water baths. This equipment is suitable for this experiment because it is easily available, it is easy to set up and use and it is easy to collect results with.
This is how the experiment will be set up
I will firstly measure the volume of protease solution by using a syringe, which will be 10cm3 and then put it into a test tube. I will then get two developed films and hook wire onto each so I am able to get them out of the tube easily. The wire will be labelled so it is easy to see which film is which. I will then put the test tube into an electric water bath, which is at a specific temperature for example 300C. I will leave it in the bath for three minutes and then put the two films into the test tube. Every 30 seconds I will check to see if the film has become transparent. When the two films have become transparent I take them out of the test tube. I then check the pH of the protease solution by getting a glass rod and dipping it into the solution and then put the solution onto pH paper.
For my preliminary experiment I set up the apparatus as above. As it was only preliminary I used one film. I chose two temperatures to put two test tubes of protease into, they were 600C and 300C. I put the two test tubes into the two different electric water baths and then after three minutes put film in each. This is how the results turned out:
Temperature of water bath/0CTest tube in water bath with no developed film/secsTime taken for film to become transparent/secsRate of reaction/ 1/secs (S-1)
This table of results indicates that when the temperature increases the time taken for the film to become transparent is less. It also shows that when the temperature increases that rate of reaction also increases until it reaches the optimum temperature.
This is what I expect will happen to the results in my final experiment.
In this experiment the independent variable will be the temperature, the dependent variable will be the time it takes for the films to become transparent and the controls are:
“Y Concentration of protease
“Y Volume of Protease
“Y Film size
The experiment should be carried out the same for each test tube and the pH should stay the same for all test tubes. The concentration of the protease solution will be 0.5% and the volume of each protease solution will be 10cm3.
The range of temperatures that I am going to use will be 300C, 400C, 500C, 600C, 700C.
If I have a temperature any higher than 700C the enzyme would most probably denature. I haven’t got a temperature any lower than 300C because it would take too long for the gelatine to break down in the time given.
In my final experiment I am going to use a syringe to measure out the volume of protease needed. A syringe is accurate enough for this experiment.
I will put two developed films into each test tube to improve reliability of my results. I will also use a stopwatch to time when I put the films into the test tube and when to check the films. The electric water baths are really easy to use and they control the variables very precisely unlike heating the test tube with a bunsen burner, as the temperature can go slightly up and down.
Whilst doing the experiment I will have my hair tied back, I will wear a lab coat and I will also wear safety goggles throughout as I am using protease which if gets into your eyes it can be dangerous.