A photographic company wants to recycle the plastic in exposed (and therefore unusable) photographic film. They will need to remove the silver compound attached to the film by the protein gelatine. They decide to use a protease enzyme called trypsin
Introduction
A photographic company wants to recycle the plastic in exposed (and therefore unusable) photographic film. They will need to remove the silver compound attached to the film by the protein gelatine. They decide to use a protease enzyme called trypsin to digest the gelatine, but want to find the best conditions for the enzyme to work to save money.
Background knowledge
ENZYMES
An enzyme is a biological catalyst that speeds up the rate of reaction. Enzymes are composed of polymers of amino acids. Enzymes are mainly located in the cytoplasm of a cell. Here they control the chemical reactions occurring inside the cells. The enzymes break down the food molecules into smaller parts so it is much easier for it to pass through the blood stream. Many enzymes are produced in different parts of the digestive system. The digestive system produces enzymes that break down the components of the food we eat.
They speed up reactions by a process called the induced fit theory. It was first thought that the 'lock and key' hypothesis was how the enzymes work, but this is now believed to be incorrect because the enzymes can change shape so that the substrate (the thing that is broken up) can fit into the active site of the enzyme, which is why it is called the induced fit theory. The lock and key theory suggests that the enzymes are the correct shape without having to change for the substrate to fit in, which they often are not. The substrate enters the enzyme and is digested in the active site of the enzyme.
WHAT IS TRYPSIN?
Trypsin is an enzyme that acts to degrade protein; it is often referred to as a proteolytic enzyme, or proteinase. Trypsin is one of the three principal digestive proteinases, the other two being pepsin and chymotrypsin. In the digestive process, trypsin acts with the other proteinases to break down dietary protein molecules to their component peptides and amino acids. Trypsin continues the process of digestion (begun in the stomach) in the small intestine where a slightly alkaline environment (about pH 8) promotes its maximal enzymatic activity. Trypsin, produced in an inactive form by the pancreas, is verysimilar in chemical composition and in structure to the other chief pancreatic proteinase, chymotrypsin. Both enzymes also appear to have similar mechanisms of action; residues of histidine and serine are found in the active sites of both. The chief difference between the two molecules seems to be in their specificity, that is, each is active only against the peptide bonds in protein molecules that have carboxyl groups donated by certain amino acids. For trypsin these amino acids are arginine and lysine, for chymotrypsin they are tyrosine, phenylalanine, tryptophan, methionine, and leucine. Trypsin is the most discriminating of all the proteolytic enzymes in terms of the restricted number of chemical bonds that it will attack. Good use of this fact has been made by chemists interested in the determination of the amino acid sequence of proteins; trypsin is widely employed as a reagent for the orderly and unambiguous cleavage of such molecules.
Below is the word equation for the process of recycling exposed photographic film with the enzyme, trypsin: -
Below is a diagram of the lock and key theory
Above is a diagram of the induced fit theory.
Aims of my investigation
In the investigation I am going to study one of the four factors that alter the rate of reaction of trypsin in the clearing of photographic film. In photographic film is the clear acetate at the bottom and on top of that is geletin (a substrate) which has silver salts in it. When trypsin is added, the gelatin is broken down and the acetate becomes clear. The reaction can occur at different speeds depending upon the factors acting on the trypsin. These factors are the pH of the environment, the temperature, the concentration and inhibitors. The factor that I will be controlling in this experiment is temperature.
Prediction
The factors that will affect the rate of reaction of Trypsin are....
pH
This can alter the rate of reaction because if the pH is too high or too low for the enzyme, it will become denatured, where the structure of the enzyme is changed so that substrates will no longer be able to fit inside the enzyme and therefore will not be digested. Most enzymes work best at pH 7, although some enzymes in the stomach work best in very acidic conditions (pH 1 or 2). The enzyme in my investigation (trypsin) works ...
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Prediction
The factors that will affect the rate of reaction of Trypsin are....
pH
This can alter the rate of reaction because if the pH is too high or too low for the enzyme, it will become denatured, where the structure of the enzyme is changed so that substrates will no longer be able to fit inside the enzyme and therefore will not be digested. Most enzymes work best at pH 7, although some enzymes in the stomach work best in very acidic conditions (pH 1 or 2). The enzyme in my investigation (trypsin) works best between pH 7 and 8, because it works in the duodenum which is slightly alkaline as pancreatic juice is present in the duodenum which is alkaline.
Temperature
Temperature can alter the rate of reaction because enzymes work best at 40°C and if the temperature is lower then the molecules will move slower and therefore there will be less collisions of enzymes with substrates and the rate of reaction will be slower. If the temperature is higher than 40°C then the enzymes may become denatured and they will not work properly, slowing down the rate of reaction or if the temperature is high enough to denature every enzyme, no reaction will occur.
Concentration
There are two ways that concentration can affect the rate of reaction, these are concentration of substrate and concentration of enzymes. The concentration of substrate can alter the rate of reaction because if it is higher then there will be more frequent collisions with enzymes and therefore a higher rate of reaction. If the concentration of the substrate is lower then collisions of enzymes and substrates will be less frequent and the rate of reaction will be slower.The concentration of enzymes affects the rate of reaction in a similar way because if there is a lower concentration of enzymes then there will be less frequent collisions and the rate of reaction will be slower. If there is a higher concentration of enzymes then there will be more frequent collisions and therefore a faster rate of reaction.
Inhibitors
These are a sort of poison that can be added to an enzyme and will block off the enzyme so that no substrate can enter the enzyme. Therefore the substrate can no longer react with the enzyme so this will certainly slow down the rate of reaction.
The factor that I will be investigating is temperature. I will see how long it takes for the photographic film to go transparent at different temperatures ranging from 10 oC - 70 oC. I predict that as the temperature increases from 10 oC - 40 oC, the faster it will take for the photographic film to go from opaque to transparent (rate of reaction of trypsin increases). As the temperature increases beyond 50 oC, the longer it will take for the film to go clear as it is entering its denaturisation process (rate of reaction of trypsin decreases). At a temperature of about 60oC the enzyme would be denatured and the trypsin will not be able to digest the protein gelatine anymore. This means that the photographic film will stay opaque.
Reasons
One of the main reasons for my prediction is the collision theory. The collision theory is based on the knowledge that chemical reactions take place by probability. In order to react, particles need to collide in the right course with enough energy to overcome the activation energy. If there are more collisions in a reacting system the rate of reaction is faster. In the reaction between the trypsin and the protein gelatine, the concentration and the temperature can affect the amount of collisions. The concentration of trypsin used will stay the same in the experiment; however there will be a range of temperatures used. As the temperature of the trypsin molecules increases they will gain more kinetic energy and therefore collide more often with the gelatine molecules causing there to be a faster rate of reaction. This is the kinetic theory, which states that as particles are given more heat, the more kinetic energy they will gain (particles move around faster). Therefore as the temperature increases, the trypsin molecules will gain more energy. This means that they will gain more kinetic energy, which will cause them to have more collisions.
Activation energy is the minimum energy required before a reaction can occur. You can show this on an energy profile for the reaction. For a simple over-all exothermic reaction, the energy profile looks like this:
The protease enzyme trypsin is located in the bovine pancreas, in the form of trypsinogen, therefore it works best at body temperature. This means that the optimum temperature for trypsin is around 40oC. After about 50oC the enzyme activity of trypsin will start to decrease. This is because it is not at its optimum temperature. Also the rise in temperature will cause the trypsin to denature and therefore slow down or stop the rate of reaction. There might not be a big decrease at 50 oC because the kinetic theory still applies where the enzyme is moving faster and colliding more often. However at around 60 oC, the denaturisation process will affect the enzyme more than the kinetic theory, meaning there will be a bigger decrease in the rate of reaction, or, the rate of reaction will stop.
A predicted graph of the rate of reaction of trypsin as temperature increases.
The graph show my prediction of what will happen to the rate of reaction of trypsin as the temperature increases. My prediction is supported by the facts that as temperature increases the number of collisions are more numerous and the activation energy can be overcome enabling the particles to react. However trypsin has an optimum temperature at where it will work best and speed up the rate of reaction. After about 50 oC the enzyme will start to denature and therefore slow down the rate of reaction. If the enzyme starts to denature then it will become useless and not be able to digest the protein gelatine as efficiently because the active site would have changed its shape and the substrate would no longer fit into it and react. The rate of reaction will have stopped by about 60oC as a result of this.
Apparatus
The following equipment will be required: -
* A kettle
* A beaker
* Ice cubes
* Exposed photographic film
* Trypsin 1%
* A splint
* A thermometer
* A test tube
* A stop watch
* A measuring cylinder
* Scissors
* Ruler
Method
) Collect all equipment and set up as shown in the Apparatus.
2) Boil water in the kettle.
3) Pour 200mls of the boiled water in a beaker. This will be used constantly in the experiment so keep it aside for future use.*
4) Cut the photographic film 1cm x 1cm using a ruler and a pair of scissors.
5) Split a splint and place the photographic film in between the split.
6) Measure 4mls of trypsin, being aware of the meniscus (measurement read at eye level), using a syringe.
7) Put the 4mls of trypsin in a test tube.
8) Put the test tube into the beaker of hot water.
9) If a temperature below 20 oC is wanted, such as 10 oC, then the beaker should be filled with ice cubes and cold water. The test tube should then be placed into this.
0) Shake the thermometer to get rid of any previous readings. The temperature should be at around 20 oC (room temperature) once shaken.
1) Place the thermometer in the test tube to measure the temperature of it.
2) Once the required temperature is reached, take the test tube out of the beaker and then place the splint with the photographic film attached to it in the test tube.
3) Start timing with a stop watch.
4) Keep the thermometer in the test tube to make sure that the temperature remains the same throughout the experiment. If the temperature begins to decrease, put the test tube back into the beaker of hot water to regain the required temperature.
5) Stir and shake the splint.
6) Take the photographic film out when the film is transparent. Stop timing.
7) Record the results in a table.
8) Repeat the experiment for a range of temperatures, such as 10 oC - 70 oC and for each temperature, repeat the experiment three times.
Safety Precautions
* Tell the teacher of any breakages.
* Do not run with equipment
* Keep containers as far away from the edge of tables as possible
* Be careful not to knock over any containers filled with hot water so no one burns themselves.
* Wipe up any water that is spilt on the floor so no one slips.
* Wear goggles to protect eyes from trypsin, as the enzyme can digest proteins in the eyes.
* Trypsin can stain clothes so take care when handling it.
* Wash hands with cold water if skin comes in contact with hot water.
* If skin comes in contact with trypsin, immediately wash hands with cold water. Do not rub eyes or touch face before you have washed your hands.
Take care when pouring hot water and trypsin into containers
The beaker of hot water may need to be replaced for certain temperatures. For example if you want to make the trypsin 70 oC and the beaker of hot water is at 60 oC, you will need to boil more water in the kettle to get the temperature above 70 oC.
The variable that I will be controlling in this experiment is the temperature, and I will use different temperatures from 10 oC - 70 oC. The dependent variable is the time it takes for the photographic film to become transparent and colourless. The controlled variables are listed below.
* The person who shakes the splint in the test tube. Different people could shake the splint more or less vigorously than another person, resulting in different rates of reaction
* The same concentration of trypsin will be used as it will be trypsin 1%. A higher concentration of trypsin could cause the rate of reaction to be faster, where a lower concentration would cause the rate of reaction to decrease.
* The size of the photographic film will always be 1cm2 (1cm x 1cm), by using a ruler to measure the axis, so that the surface area is the same for each experiment and the trypsin in each one have the same amount of substrate to digest.
* The pH level will not be changed as different levels could change the performance of the enzyme by either enhancing it or denaturing it. It is known that the optimum pH for trypsin to work is 7-9. The pH will be kept neutral at 7.
* The stop watch will be kept the same so that the time is recorded from the same source for each experiment. Different stop watches could have different time delays so it is better to use the same stop watch for each experiment.
* The person who judges whether the film has become transparent as different people have different levels of vision and one person may think the film had gone clear, but another does not. Therefore the same person should be the judge.
* The thermometer used as different thermometers may give different readings. In order to have accurate results, the thermometer used has to be the same.
* The volume of trypsin will remain the same by measuring the amount with a syringe. This will make sure that there is no vary in concentration of trypsin, where an increase in concentration can cause the rate of reaction to speed up.
RESULTS
In the table below are the results that I gathered.
Temp?C
Time (1) seconds (minutes)
Time (2) seconds (minutes)
Time (3) seconds (minutes)
Average time seconds (minutes)
Average ROR seconds
0
N/A
N/A
N/A
3600+ sec (60+ min)
0.00028000+
20
264 sec (21:04 min)
368 sec (22:48 min)
291 sec (21:31 min)
308 sec (21:48 min)
0.0007645
30
830 sec (13:50 min)
853 sec (14:13 min)
838 sec (13:58 min)
840 sec (14:00 min)
0.0011905
40
91 sec (3:11 min)
85 sec (3:05 min)
96 sec (3:16 min)
91 sec (3:11 min)
0.0052356
50
289 sec (4:49 min)
272 sec (4:32 min)
279 sec (4:39 min)
280 sec (4:40 min)
0.0035714
60
391 sec (6:31 min)
331 sec (5:31 min)
363 sec (6:03 min)
362 sec (6:02 min)
0.0027624
70
N/A
N/A
N/A
N/A Trypsin denatured
N/A
Conclusion
The graphs on the previous pages show that the trypsin works best when it is at 40?C, exactly what my prediction stated. My average time against temperature graph clearly shows this. My prediction that after 40?C the optimum temperature for the trypsin to work in is left further behind and the trypsin begins to denature finally becoming fully denatured at 70?C. Overall my predictions were correct. The amount of collisions occurring increased as the temperature increased. My other graph that shows the average ROR, agrees with my prediction that the trypsin was the most effective at 40?C (the temperature the enzymes work best in) because the ROR after that fell as the trypsin denatured and the active site would have changed shape and therefore the substrate would no longer fit into it and react so the average ROR decreased. The ROR was increasing because of more kinetic energy causing collisions and faster reactions. One of the things that was different from my prediction was that the Trypsin would become denatured earlier than it actually did. All these findings are explained by collision theory so my predictions were correct.
Both of these graphs show the activation energy with an enzyme and without an enzyme. I can clearly relate the graph with an enzyme to my results of my experiment. This relates because the enzyme trypsin reduces the need for activation energy and so it allows reactions to take place more readily and at lower temperatures than would otherwise be necessary.
I had one slightly anomalous result that I will explain during my evaluation.
Evaluation
Overall I believe my results are accurate enough to support a firm conclusion however there things that could be done to improve the accuracy of the experiment. The size of the film was not all ways the same, so if you put two films in two different test tubes in water baths at the same temperature, then you might get one film transparent before the other even though everything else is the same. This is because the surface area of the film that is slower at turning transparent compared to the other film that was transparent before is greater and this doesn't make my results 100% reliable. A reason for my anomalous result could be that as the Trypsin was produced on 24/9/02 and was used on 3/10/02 when we opened the flask which contained trypsin, we left the lid open so it is left to be contaminated by the infectious environment (Bacteria or germs). So this might be the reason why the reaction was slower than expected at 30?C.
Learning from this the overall improvements I could have were...
* to keep the lid on the flask containing trypsin, use a light on one side of the test tube and a light sensor on the other side of the tube connected to a stopclock so when the light sensor detects light through the acetate when it is clear, it would stop the clock (this would eliminate the problem of human error in stopping the clock at an incorrect time)
* measure all the film pieces at exactly the right size for a fairer and more accurate result
* make sure the trypsin was fresh and cold
* try different types of photographic film
* conduct the experiment at more temperatures for example 35 degrees 45 degrees etc.
* use an electronic temperature probe
Extension
I could do an extension by controlling a different factor while doing this experiment like the size of the film or the concentration of the trypsin. The experiment could be repeated but instead of measuring the time against temperature it could be measured against the concentration. The film would be dropped into different concentrations of trypsin in a controlled environment and the time measured for the film to become clear measured.