Throughout this experiment I will be using the same buffer solution provided, and will not make any changes of my own that might affect the enzyme activity.
pH is a measure of hydrogen ions in a solution. The greater the concentration of hydrogen ions the lower the pH.
Most enzymes function at a narrow pH range. A change in pH may create a difference in the enzyme activity.
The pH of trypsin is between 8 and 9 and the pH of buffer solution is 9.
The pH of the buffer solution is very near to the pH of trypsin, which means that the enzyme will work at its optimum pH.
The greater the enzyme concentration the less time it takes for casein to dissolve. The enzyme trypsin will break down the casein molecules and allow it to become soluble.
If there are more enzymes present/greater concentration, there will be more active sites available for the substrates to fit into allowing more products to be formed.
When the substrate concentration is maintained at a high level and other variables such as Temperature and pH are kept constant, the rate of reaction will be directly proportional to the enzyme concentration.
Affects of pH on the Enzyme Activity
- Changes in pH may lead to the breaking of ionic bonds that hold the tertiary structure of an enzyme in place. The enzyme begins to lose its functional shape, specifically the active site.
- The substrate molecule will no longer fit in the enzyme and the enzyme is said to be denatured.
- Moreover, the changes in the pH affect the charges on the amino acids within the active site. The enzyme will be unable to form enzyme substrate complexes.
Apparatus
- Casein 5 cm³ (2%)
- Trypsin 5 cm³ (1%)
- Measuring Cylinder 5 cm³
- Test Tubes
- Test Tube Holder
- Pipette
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Distilled Water (as required)
- Stopwatch
- Goggles
Method
The most important procedure takes place at the beginning of the experiment when the apparatus is thoroughly cleaned with distilled water, to remove unwanted substances that may create differences in the set of results.
This experiment will be carried out at room temperature approx. 23°C; therefore every test will experience equal intensity of heat.
Firstly, I will measure 5 cm³ of casein in a measuring cylinder. This procedure will be repeated with the enzyme called trypsin. However for trypsin, there will be a particular concentration.
I will make use of the pipette by adding or removing few drops of the solution until they reach the required volume. This step will contribute to my set of results by improving the accuracy.
In a test tube, trypsin will be added to casein and I will start the stopwatch. The time will be recorded for the duration of the solubility of casein. A colorimeter can be used to accurately judge when all the casein has been dissolved. This method will be repeated with the other enzyme concentrations of trypsin.
Since I am only provided with one concentration of trypsin (1%), I have to prepare other trypsin concentrations by combining different volumes of (1%) of trypsin with distilled water.
Each enzyme concentration will be tested 3 times, which will improve the accuracy in calculating the average time it takes for casein to dissolve.
Safety
Safety goggles must be worn at all times throughout this experiment to prevent any hazardous chemical solutions such as casein or enzyme trypsin from entering the eye.
- Interpretation of Results
I have completed the practical part of this assignment and have entered the collected data into my table of results. By examining the set of results I have acknowledged a certain pattern to appear between the time taken for the saturation of casein, with the concentrations of enzyme trypsin solution.
The greater the concentration of enzyme trypsin solution, the less time it will take for the casein solution to clear.
- Evidence from the Collected Data
My set of results show a very similar correlation between the enzyme trypsin concentration against the time taken for the casein solution to clear. The enzyme trypsin will break down the casein molecules and allow it to become soluble and transparent.
By increasing the enzyme concentration there will be more enzymes present; there will be more active sites available for the substrates (casein) to fit into allowing more products to be formed in a given time. The bonds between the casein molecules will be broken even more rapidly allowing the solution to become clear.
When the substrate concentration is maintained at a high level and other variables such as Temperature and pH are kept constant, the rate of reaction will be directly proportional to the enzyme concentration.
After plotting the appropriate data onto a graph I have found one anomaly.
This anomaly has appeared on (1/Time against Trypsin Concentration) graph.
It’s precise point…
Anomalies typically occur due to experimental errors. During this experiment there were possibilities of coming across different errors which could have been related to the apparatus and procedures involved in this practical.
- Some of the main possibilities which create anomalies…
- Even though the apparatus, such as the test tubes were cleaned thoroughly with distilled water, every time they were used, there might have been some droplets of another unknown solution which were not seen. Those drops of liquid may have effected and caused variation in the set of results.
- The solutions enzyme trypsin and casein were involved in this experiment. These solutions were buffered. Buffer solutions are known to maintain a constant pH. Throughout this experiment I have used the same buffered solutions provided to me. There might have been few errors in making the buffer solution which may have caused changes in the pH and effected the collection of data.
- Measuring the solutions can also be an important factor. I have used the correct measuring apparatus, such as the measuring cylinders and pipettes, all to their correct scale and to the appropriate requirement, for accurate results. However, there could have been precision errors. Even though I had measured the exact amount of the solution needed, by looking at the meniscus, errors still may occur and affect the overall results.
- When the reaction is underway and the casein solution is beginning to clear away, it can be very difficult to point out when the reaction has come to an end. This is because sometimes it may seem that the casein has cleared, but in fact some of its residue are still present. The time recorded can have an effect on the average timings and the other of the outcomes.
- To reduce or eliminate errors…
- Thoroughly cleaning all the apparatus which will be involved in the experiment with distilled water. Additionally, use particular tools if necessary to clean the apparatus and making sure it doesn’t get damaged.
- The solutions of casein and enzyme trypsin must be prepared on the day of the experiment and must be buffered at the correct pH at which the enzyme can react with the substrate. This will surely improve the accuracy of the collected data.
- Making sure that the correct apparatus is used to measure all solutions. When measuring casein or any other solution make sure that there is no air in the pipette, because air bubbles will form making it difficult to read off the scale. Moreover, apply the meniscus method to read off the apparatus.
- When measuring different solutions, make use of separate measuring cylinders for each solution, to prevent contamination and inaccurate results.
- Test the solubility of casein with the enzyme trypsin by adding equal amounts in a test tube. Shake the test tube once at the beginning of the test and commence the reaction by starting the stopwatch. You may shake the test tube at intervals to speed up the reaction. Carefully watch the reaction and stop the stopwatch when you think the solution has become transparent and all the casein has dissolved.
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Furthermore a colorimeter can be used to accurately judge when all the casein has been dissolved. It is an apparatus that characterizes the colour samples to provide an objective measure of colour characteristics. The colorimeter allows the absorbance of a solution at a particular frequency of visual light to be determined. A more concentrated solution will give a higher absorbance reading.
- Sources of error on the reliability of results…
All experimental uncertainties are either due to Random or Systematic
Errors. Systematic and Random Errors refer to problems associated with
making measurements. They are mainly related to the measurement of
solutions involved in this experiment.
Random errors are statistical fluctuations in the measured data due to the precision limitations of the measurement device. Random errors can be evaluated through statistical analysis and can be reduced by repeating the experiment a number of times and averaging over a large number of observations.
Systematic errors are inevitable and produce inaccuracies that are similar and consistently in the same direction. Systematic errors are often problems which continue throughout the entire experiment.
Systematic errors are difficult to detect and cannot be analyzed statistically, because all of the data is off in the same direction. It is either to high or too low. Spotting and correcting for systematic error takes a lot of care.
- Other Experimental Errors and Uncertainties
I believe that the pH of the buffered solutions such as casein/trypsin
changed and was unable keep the pH constant. Therefore the enzyme
could not resact with the casein molecules.
It is also very difficult to judge when all of the casein has become soluble.
There will always be a difference in the timings recorded. Therefore, a
colorimeter can be used to accurately judge when all the casein has been
dissolved.
It is an apparatus that characterizes colour samples to provide an
objective measure of colour characteristics. The colorimeter allows the
absorbance of a solution at a particular frequency of visual light to be
determined.
The way to achieve the most accurate results is to use the most recently
made buffered solutions, measuring them using the correct and clean
apparatus, allowing the reaction to take place in a clean test tube washed by
distilled water and record the time it takes for the casein solution to clear by
holding it up to a bright light and verify transparency.
