An investigation to determine the effect of concentration of protease, trypsin has on the rate of digestion of protein.

Enzymes work best at their optimum temperature, for human enzymes, this is around 37˚C. I do not want the temperature to be so low that the reaction would be very slow, nor do I want the temperature to be so high that trypsin I used would be de-natured. Therefore I decide to set the temperature at 35˚C. Temperature will be kept constant by the water in the beaker(temperature of water needs to be maintained at 35˚C). If the temperature decreases, then I can add warm/hot water in from the kettle, if temperature rises, then small amount of ice cube can be added to lower the temperature.

• The volume of trypsin and milk used for each experiment will need be constant. Volume of solution is measured by a 20cm3 syringe, which gives an accuracy of ±0.5 cm3.
• The concentration of protein solution needs to be constant, because it affects the chances of particles colliding with each other, which affects the rate of reaction. I can control the concentration of protein solution by using milk powder. I can either use the same solution I made for experiment, or mixed the same amount of milk powder with the same amount of water for each experiment.
• The ph of the protein solution would need to be kept constant, as trypsin works better at a specific ph. Most enzymes are sensitive to ph levels and can become denatured by too low, or high ph. Trypsin is not an exception, it only works within a certain range of ph levels. The ph should be kept the same, by the use of a buffer, at its optimum level, which is around 7(according to the AS biology test book specification A).

• The size and thickness of the cross that is used to stick on the test tube containing protein solution would need to be the same for every single experiment I do.

• The only variable I am changing is the concentration of the enzyme (trypsin). The turnover number of Chymotrysin is about 6000, which means that a trypsin molecule is capable of converting reactants into 6000 products in a minute. There are many different type of trypsin and their turnover number may vary, but it should be around 6000. As the turnover number is fairly high, in order to see the affect of different concentration has on the rate of reaction, the ranges of concentration that I use for my investigation will need to be very low i.e. below 1%. I decided to use 0.001, 0.005, 0.01, 0.05, 0.1 and 0.5 for my concentration ranges.

Method:

• Safety glasses, apron and disposable should be worn at all times during experiments
• Set up the experiment as shown on the diagram, but do not pour any solution in any equipment yet.
• Boil water, then pour it into a beaker and wait until it drops to around 30˚C. Ideally, the bigger the beaker the better, as a bigger volume of water helps in maintaining the temperature at the chosen temperature.
• I have decided to use 20cm3 of protein solution and 4cm3of trypsin to carry out my experiment.
• Suck 4cm3of trypsin (of different concentration each time) using a 20 cm3 syringe.  This gives a degree of accuracy of ±0.5 cm3.
• Measure 20cm3 of protein solution using a 20 cm3 syringe, again, this is accurate to ±0.5 cm3
• Put the measured protein solution in a test tube.
• Put the syringe, containing trypsin, and the test tube containing protein solution in the beaker for around 2 minutes for them to reach 30˚C. (Use the thermometer to measure the temperature).
• Print off several crosses and stick one on the test tube containing the protein solution.
• Add the measured trypsin into the protein solution and start the stop watch simultaneously after 2 minutes.
• The cross should not be visible through the solution at the moment.
• When the protein has digested by trypsin, the colour of the solution will be clear and I will be able to see the cross through the solution.
• Look at the cross through the test tube.
• Stop the stopwatch when the cross can be seen.
• Repeat experiment for each concentration of trypsin three times and take the result of the average. This can make the result more reliable.