These form tempary bonds with the amino acids forming an enzyme – substrate complex. During each separate reaction products are formed which are released and enzyme concentration remains the same. The enzyme works only for a specific reation due to its uniquely shaped active site. Once the substrate is in the active site the molecule changes shape around the substrate. Energy is needed in the reation to start it and to break chemical bonds to convert the substrate into products; this energy is called the activation energy.
At the beginning of the reaction there are many enzyme and substrate molecules, therefore many active sites are open for reaction. The greater the concentrations of protease, more active sites become available to casein substrate molecules so the rate of reaction is quicker. As the reactions take place active sites become occupied rapidly increasing the rate of reaction. Over time increasingly less protease are being used as casein molecules are used up gradually decreasing the ate of reaction. This is showed in the diagram (1):
Diagram 2 shows the effect of enzyme concentration on the rates of reaction. The rate of reaction is directly proportional to enzyme concentration because of the increasing number of active sites available.
Plan
The apparatus I am going to use for the experiment include:
- Clock
- Ruler, pen
- Testubes/chronical flacks
- Acidified protease solution
-
5cm3 pipettes
- measuring cylinder
-
50cm3 beaker
- glassware to dilute enzyme milk powder solution(substrate)
- testube holder
- thermometer
These apparatus are appropriate and suitable for making accurate measurements because they measure to a good degree of accuracy. The pipettes measure to 0.2cm3 with individual drops of 0.05cm3, the measuring cylinder measure to 0.1cm3 and also the clock measures to 1/100ths. The thermometer measures to 10oC.
Method
-
Organise 5 solutions of 5cm3 each containing different concentrations of protease solution. The different concentrations can be achieved by mixing different quantities of 1cm3 of distilled water with the acidified enzyme protease. The 5 different concentrations of enzyme solution will include 1cm32cm33cm34cm 3 and5cm3.
- Now 5 testubes need to be filled with exactly the same volume of 5cm3 of milk powder substrate solution containing the protein casein.
- Pipette each 5mc3 enzyme concentration solution into separate substrate solutions.
- Start the stop clock immediately and record the time taken up to the point when the milk powder protein solutions clear.
- Repeat the procedures 3 times so that consistent results can be obtained for a suitable average result.
Independent variable
The independent variable is the concentration of acidified protease enzyme used. The different concentrations used will be:
These different concentration levels will be accurately changed measuring xcm3 of enzyme and xcm3 of distilled water to the appropriate ratio of volume each and mixing them together. Each enzyme solution contains an exact volume of 5cm3.
Dependent variable
The dependent variable involved is the time taken for all the casein to be broken down clearing the milk powder solution. The time will be measured immediately after the protease has been completely added to the milk using a stop clock measuring to 1/100ths. I can ensure accurate results by keeping all the other factors like temperature constant to make it a fair test. Also a repetition of results will be taken so average results can be obtained from them which will make the overall results more reliable.
Other variables
Other variable include temperature, concentration of substrate and volume of solutions. A thermometer will be used so that tests will only be started when temperatures are the same. If temperature is not monitored the enzyme activity will alter increasing or decreasing. The temperature should not vary too much as the experimental conditions and surroundings will be the same as the experiment will be carried out in a controlled environment. The substrate concentration will be kept the same for each test to ensure fair tests. If the concentration is increased then the time taken for the casein to be broken down will increase as more substrate molecules will be present.
Risk assessment
This experiment is a fairly safe one however there are acidic chemicals involved which can cause irritation to skin and burn. Therefore eye protection and protective clothing is highly advised and no chemicals should ever be consumed.
Results
Graph
X-axis- acidified protease enzyme concentration (%)
Y-axis- time taken for the milk solution to clear (seconds)
Trends and result analysis:
By looking at the results obtained and the graph they both show evidence proving the hypothesis that;
‘Substrate solution will clear quicker with a higher enzyme concentration’.
The lowest concentration of 20% cleared very slowly and showed a major difference in time to clear compared to the 40% where there was a significant drop on the graph. From then on there was almost a constant difference with time variable illustrated by the considerably straight line from 40% to 100%. The reason for the gradual increase in time is that many of the active sites available were becoming rapidly occupied. As time increased less protease was being used as continuously less casein molecules need breaking down, therefore gradually decreasing the rate of reaction.
However the 20% represents an anomalous result because the time taken was not in proportion to the other results. This could have been caused by over diluting the protease making it less reactive or by adding too much of the substrate increasing the amount of reactions to take place.
Conclusion:
In conclusion to this experiment I am very pleased as it was carried out well and accurately apart from one anomalous result. Many improvements can be made to the experiment to make it more reliable and accurate. Specialised equipment would be an appropriate option to make all measurement more accurate, eg, to measure the volume to 0.01cm3 instead of 0.1cm3. I can calculate the %error of the volume measurement to see how accurate it was:
%error = 0.1cm3 X 100 = 2%
5cm3
This shows that the volume of the solutions was measured very accurately, however 10X more precise equipment would lead to just a 0.2% error which is extremely good.
Also repeating the experiment many more times, taking 10 trials instead of 3 will illustrate results more accurately and analysis would be more critical. Finally to improve the experiment I would use a much wider range of % concentrations adding to the reliability of the experiment results.