Enzymes are also affected by changes in acid and alkaline conditions. Some enzymes work in acid conditions whilst others in alkaline conditions. Most enzymes work best at a certain level of acidity or alkalinity. The optimal pH for an enzyme depends on its site of action. For example, enzymes in the stomach have an optimum pH of about 2 because the stomach is acid, but intestinal enzymes have an optimum pH of about 7.5. The graph on the left shows how enzymes work at different pH levels.
The enzyme we will be working with is Trypsin. Trypsin is a proteolytic enzyme formed in the intestine or pancreas of an animal. Trypsin breaks down arginine or lusine and works only in an alkaline setting.
VARIABLES
FACTORS THAT I CAN VARY IN THE EXPERIMENT:
- Concentration of Trypsin
- Concentration of milk
- The pH of solution
- Temperature
FACTOR THAT I CAN MEASURE:
- Time taken for enzymes to digest the casein in the milk
PRELIMINARY WORK:
I have decided to change the concentration of the Trypsin in my experiment. This is because after some preliminary investigation, I found out that it was too hard or complicated to try to change any other of the variables instead.
In each experiment I will use 10cm³ of milk and 5cm³ of trypsin.
These are the concentrations I will be using:
I also decided that I would justify that the experiment is over, and the casein in the milk has been digested when a black dot can be seen from behind it i.e. the milk becomes translucent.
METHOD
Apparatus:
- 1% Trypsin solution
- 4% milk solution
- Test tubes
- 10cm³ and 25cm³ measuring cylinders
- Distilled water
- Plastic pipettes
- Test tube rack
- Stopwatch
- Piece of paper with a very clear black dot
Procedure:
Firstly the desired concentration of the trypsin was made by diluting the trypsin with the distilled water as shown in the table above. Then exactly 10 cm³ of milk was measured out into a test tube and placed on a test tube rack. Then, making sure that the stopwatch was started at the same time, the trypsin solution was added to the milk, and then with a thumb over the top of the test tube, it was shaken exactly 10 times. It was then placed back in the test tube rack. Now the piece of paper with the black dot was placed behind the test tube, and when this dot became visible, the stopwatch was stopped and the time was noted down. This procedure was then repeated with all of the different concentrations.
Fair Testing:
- Make sure the same dot is used as an ending point in all experiments as if another dot is drawn; it could be more, or less visible than the last.
- Make sure that the end point is fairly determined – As soon as the dot can be seen at least a little bit, the experiment should be stopped.
- Make sure the same person does the shaking of the test tube and he shakes it with the same strength in each experiment.
- Make sure that only distilled water is used, because tap water may contain impurities, which can affect the results.
Safety:
- Be careful to not splash any of the solutions as it can be very irritable if the trypsin gets into somebody’s eyes
- Other than that, follow the basic simple classroom rules
PREDICTIONS
After carrying out my preliminary studies and also after studying some textbooks, I have been able to make a prediction:
As the concentration of the trypsin is doubled, the rate of digestion of the casein in the milk will also double.
Therefore, the rate of the digestion of the casein in the milk is proportional to the concentration of the trypsin.
This is what I expect the graph showing rate of digestion, against the concentration of the trypsin solution to look like:
0
Conc. of trypsin (%)
I am expecting this because when the concentration rises; there will be more trypsin molecules in the solution. Therefore more trypsin will come into contact with the casein in the milk, there will be more reactions, and so it will digest the casein in the milk and turn it translucent more quickly.
From this graph I can see that as the concentration of the trypsin solution gets higher, the time taken to digest the milk becomes less. The line is straight and goes through the origin (0,0). From examining the graph, I can see that when I double the concentration of the trypsin, the rate of digestion also doubles.
To further analyse my results, and now I know that the graph shows direct variation, I am able to work out a constant by which the factor increases:
The general algebraic direct variation equation is as follows:
X ∝ Y (where X and Y are different factors)
The sign ∝ symbolises that X varies with Y. For example in this investigation:
X = Concentration of trypsin
Y = Rate of digestion
If X ∝ Y
Then X = K x Y (k = constant)
I will take a point in the line, and read off X and Y (see *1 on the graph)
(X= 0.5, Y= 0.0037)
X = K x Y
0.4= K x 0.0037
∴K= 0.5
0.0037
K= 135.14
∴X = 135.14 x Y
and Y = X
135.14
Now I will prove that the constant I have calculated is correct.
I will work out the rate of digestion when the concentration of the trypsin is 0.2%
X = 0.2%
Y = X ÷ 135.4
Y = 0.2 ÷ 135.4
Y = 0.0014
The answer is correct (See *2 on the graph)
The reason for there being this trend between the two factors is as I previously described in the Planning section:
When the concentration rises, there will be more trypsin molecules in the solution. Therefore more trypsin will come into contact with the casein in the milk, there will be more reactions, and so it will digest the casein in the milk and turn it translucent more quickly. When the concentration of the trypsin solution is low, there are less actual trypsin molecules in the solution and therefore there will be fewer reactions and so it would take more time for the milk to be digested.
My results did actually agree with my predictions. Even though there were quite a few anomalies, I was able to draw a straight line through the origin.
Even though the results are not completely accurate, I can still draw a firm conclusion to my experiment:
As the concentration of the trypsin is doubled, the rate of digestion of the casein in the milk will also double.
Therefore, the rate of the digestion of the casein in the milk is proportional to the concentration of the trypsin.
There were quite a few reasons for why my results may have been inaccurate:
- It was very difficult to measure out the exact amount of trypsin in the measuring cylinders. This is because we were using such small amounts of trypsin.
- It was very hard to make sure that the trypsin-milk solution had been evenly shaken in each experiment as we were just using our hands.
- It was hard to determine when the milk had been digested because we were only using our eyes to see how translucent the solution had become.
- As the experiments were performed over a period of a couple of lessons, the room temperature would have been different each time. This could have dramatically changed the speed of the reaction as the higher the temperature, the faster the rate of reaction.
Seeing as all these problems were faced during the experiment, I think the experiment was done as accurately as it possibly could have been done. There were quite a few anomalous results. This may have been because the temperature was especially high, or low on those days and therefore the reaction went faster or slower than it should have done.
Given the conditions, and the problems we faced that could not have been helped in any way, the experiment was as fair as it could have possibly been.
The accuracy of the results could have been improved by doing the whole thing in one day. This way, the temperature would have stayed relatively the same. Or even better, a water bath could have been used. This would mean that the temperature would stay the same throughout the experiment. To make sure that the measurements of the solutions were, correct, a burette could have been used, which is very precise and would have made the results more accurate. Also, to decide when the experiment was over a light detector could have maybe been used; a would have been shone through one side of the test tube, and when the milk has been digested and it turns translucent, the light detector would indicate this, and my results would have been more accurate. Also, it may have also been a good idea to use some type of stirring device so that the results would have been a bit fairer.
The experiment could have been extended by varying the temperature in which the reaction took place. Or I could have also varied the concentration of the milk, and also the pH of the solution. This would have helped me get a more overall understanding of how the enzyme, trypsin works.
Even though the results are slightly ‘off’, I still think I can come to a firm conclusion with them.
