Acid or alkaline solutions alter the chemical properties of proteins, which includes enzyme. Most enzymes work best at a certain level of acidity or alkaline (pH). The protein digesting enzymes in your stomach of example, they work best at pH of 2. At this pH, the enzyme amylase, from your saliva cannot work at all. Inside most cells the enzymes work best at a pH of 7, as it is neutral. The pH or temperature at which this enzyme works is called its optimum pH or temperature.
Although changes in pH affect the activity of enzymes, these effects are usually reversible, i.e. an enzyme, which is inactivated by a low pH, will resume its normal activity when its optimum pH is restored. Extremes of pH, however, may denature some enzymes irreversibly.
Structure of an enzyme:
The site on an enzyme, where the substrate is converted to a product is called the active site. It has a specific shape, which is complementary, to the shape of the substrate. The role of the active site, that the compound on which an enzyme acts (substrate) must combine in some way with it before catalysis can proceed is an old idea, now supported by much experimental evidence. The combination of substrate molecules with enzymes involves collisions between the two. Enzymes are large molecules, the molecular weights of which (based on the weight of a hydrogen atom as 1) range from several thousand to several million. The substrates on which enzymes act usually have molecular weights of several hundred. Because of the difference in size between the two, only a fraction of the enzyme is in contact with the substrate; the region of contact is called the . Usually, each subunit of an enzyme has one active site capable of binding substrate. The characteristics of an enzyme derive from the sequence of amino acids, which determine the shape of the enzyme (i.e., the structure of the active site) and hence the specificity of the enzyme.
Pilot Study
Apparatus
For this experiment I have been provided with the following apparatus:
1% trypsin solution
4% milk solution
Test tubes
10cm³ and 25cm³ measuring cylinders
Distilled water
Buffer solution, pH 2, 4, 5, 7, and 11
Plastic pipette
Water baths at 40°C and 60°C
Stopwatch
Method
Once I had received all the apparatus mentioned above, I poured 5cm³ of milk into 2 test tubes. I then drew a black ‘X’ on a piece of paper, which would show me when the solution became translucent as there being a dark background to visualise the solution becoming translucent more accurately, so when I could see the ‘X’ I would stop the time and record how long it took it took the solution to become translucent. I then added 5cm³ of trypsin into the 2 test tubes, and then immediately placed one test tube into the 40°C water bath and the other test tube into the 60°C water bath. I then timed how long it took for the solution to make the ‘X’ appear clearly. I then decided to recorded how long it took for the solution to become translucent at room temperature.
I then decided to use the pH variable. I added the five-pH solutions into the five test tubes, and then added 5cm³ of casein milk into each of the five test tubes. I then added 5cm³ of trypsin into each tube. I would then record the results, which would measure which pH solution had the most effect on the enzyme.
Results
Observations
This experiment showed that using the temperature, as a variable would be most effective rather than using the pH as a variable. The temperature near to the optimum temperature worked best, which was the 40°C water bath, and the temperature that worked the least best was the room temperature. The 60°C water bath had the same rate of reaction as the 40ºC water bath. Using pH as a variable showed that the optimum pH was 7 where the enzymes worked best.
Conclusion
I found out that the water bath that was set to 60°C, took the least amount of time to become translucent, whereas the 20°C water bath took double the time of the 40°C and the 60°C water bath. This was because the enzymes that were placed in the 20°C did not function as well as the other temperatures. The enzymes at room temperature took the longest amount of time to become translucent because the room temperature was below the optimum temperature, and enzymes work best at their optimum temperature, but in this case the enzymes did not work to their best therefore taking the most amount of time to become translucent. I realised that using the pH, as a variable would be unfair because as you add the casein milk with pH, the solution becomes translucent, without even adding the trypsin, therefore there would be no point in adding the trypsin, defeating the whole objective of the investigation. The buffer coagulates the casein and then it precipitates to the bottom of the test tube.
Predictions
I can say that the closer the temperature is to its optimum temperature, the shorter the time taken for the solution to become translucent will be. Whereas, those temperatures that exceed the optimum temperature, the enzymes will become denatured. The temperature is directly proportional to that of the rate of reaction, up and until the optimum temperature, when the enzymes become denatured and the relationship stops. If the temperature is relatively low then the enzymes do not work as well. So I can say that those temperatures that are near to the optimum temperature will take the least amount of time for their solution to become translucent. I predict that the optimum temperature will be 50°C, and after the temperature has exceeded this, the enzymes will become denatured.
Temperatures as low as 20°C and did not generate enough heat for the enzyme to catalyse properly, therefore taking a longer time for the solution to become translucent, causing a slower rate of reaction. The temperatures 40°C and 60°C had the same rate of reaction showing that every 10°C there is a change in the function of the enzyme, meaning that the enzyme works differently, as it gets closer to the optimum temperature. In the early part of the experiment the statement that made in the ‘Background Knowledge’ that ‘a rise of 10°C will double the rate of reaction’ was true for the temperatures 20°C where the rate of reaction doubled from 0.008 to 0.016, but afterwards this statement collapsed.
Method
I was given the following apparatus; 1% trypsin solution, 4% milk solution, test tubes, 10cm³ and 25cm³ measuring cylinders, distilled water, buffer solution, pH 2, 4, 5, 7, and 11, plastic pipette, water baths at 40°C and 60°C, and a stopwatch.
I firstly measured 10cm³ of casein milk with a measuring cylinder and then poured 5cm³ into two test tubes by using a measuring cylinder that were going to be placed into each water bath. I then drew a black ‘X’ on a piece of paper, which would show me when the solution became translucent, so when I could see the ‘X’ I would stop the time and record how long it took it took the solution to become translucent. I then measured 10 cm³ of trypsin with a measuring cylinder and then poured 5cm³ into the two test tubes by using a measuring cylinder. I then immediately placed one test tube into the 60°C and placed the other into the 40°C water bath. I then timed how long it took each solution to become translucent by using a stopwatch. I then poured 5cm of casein milk into an empty test tube with a measuring cylinder and then added another 5cm³ of trypsin by using a measuring cylinder. I then used a stopwatch to time how long it took for this test tube to become translucent. I could tell when the solutions became translucent when I placed the black ‘X’ behind the tube, and if the ‘X’ were seen, then I would stop the timer and record the time taken to for the solution to become translucent. I decided to calculate an average rate of reaction to make the results more accurate, because in some cases one experiment is not enough as many factor, for instance human error might affect the result, so I felt it would be best to calculate an average rate of reaction.
Results
Analyse
From the results shown on the scatter graph I can say that the optimum temperature was 50°C. The results table shows that after the temperature increased from 50°C the time taken for the solution to become translucent, increased as well, therefore causing the rate of reaction to increase. The temperature of 50°C had the greatest rate of reaction for the experiment; therefore the optimum temperature was 50°C. Temperatures such as 70°C and 80°C had no result because the solution remained cloudy, therefore the enzyme had become denatured and could not function properly. Temperatures as low as 20°C and 30°C did not generate enough heat for the enzyme to catalyse properly, therefore taking a longer time for the solution to become translucent, causing a slower rate of reaction. The temperatures 40°C and 60°C had the same rate of reaction showing that every 10°C there is a change in the function of the enzyme, meaning that the enzyme works differently, as it gets closer to the optimum temperature. In the early part of the experiment the statement that made in the ‘Background Knowledge’ that ‘a rise of 10°C will double the rate of reaction’ was true for the temperatures 20°C to 30°C where the rate of reaction doubled from 0.008 to 0.016, but afterwards this statement collapsed.
After 60°C the enzymes became denatured because the shape of the enzyme had changed and therefore cannot combine together, i.e. the lock and key theory, with the substance. The organisms are killed by the prolonged exposure to high temperatures. The enzymes in their cells become denatured and the chemical reactions happen too slowly to maintain life. If the temperature is too low, i.e. below 40°C then the enzymes have less kinetic energy so they are less likely to collide with the substrate and catalyse the reaction.
The graph shown has no anomalous results, therefore my predictions are correct, as all the results apply to what I have predicted. The only prediction that was not entirely correct was that the temperature is not directly proportional to the rate of reaction, but in the early part of the experiment this statement is true, but then the relationship breaks up, but this relationship does not matter because of the optimum temperature. The optimum temperature signifies that those temperatures either above or below do not have enough kinetic energy to function properly. The results after 30ºC are not directly proportional, so therefore I can say that the temperatures above 30ºC have anomalous results.
I can conclude that the optimum temperature for this investigation is 50°C, where the enzymes have the right amount of kinetic energy and the right shape to combine with the substance, therefore having the fastest rate of reaction.
Evaluate
The experiment could have been improved by using test tubes with pre-marked surfaces so that we could see more easily when the solution became translucent. Deciding when the solution became translucent was difficult, which causes the results to be imperfect. Implementing more time to carry out the experiments with more care and precision could have carried out the experiment better. To record the results I had to do two things simultaneously, I had to decide whether the reaction had occurred as well as checking the time taken for the reaction, which was very difficult. The trypsin warmed in the water separately could have saved more time and the results would have been better, but the trypsin would also have to be left in the water bath as its temperature decreases rapidly. When the trypsin is at the correct temperature, it would have been advisable to warm up the milk at the same time. This would make sure that the two separate solutions are not mixed, therefore allowing us to time the experiment more accurately because otherwise if the solutions were mixed there would have been a human error with the experiment, because this allows time for the substrate and the catalyst to mix together, without actually timing the experiment, making the results imprecise.
I could then have varied the concentration of trypsin, which would inevitably find out the optimum concentration for the fastest rate of reaction. I can predict that the greater the concentration, the faster the rate of reaction. I could of also have heated the test tubes separately, which I did not do for this particular experiment. If I had more time I could have done each temperature several more times and worked out an average, because the more results obtained the better the result would be and also the results would be more precise as well. Also I could have measured the milk and the trypsin more accurately, as sometimes I spilled the solution or I had to measure the solution quickly due to the restriction in time. I could alter the weight of the milk powder, and investigate how much powder is needed for the fastest rate of reaction.