Skim-milk will be used rather than whole milk because skim-milk provides more protein and therefore more casein in the experiment.
The effect of a change in PH on enzymes is the alteration in the ionic charge of amino acids at the active site, so the active site changes and the enzyme can no longer form enzyme-substrate complexes. To test the effect of various PH on the digestion of casein I decided to use PH 4, PH7 and PH9.5 because I could then, compare the effect of acidic neutral and alkaline conditions on the digestion catalysed by trypsin.
Adding buffer would maintain the PH in each experiment and results that are more accurate would be obtained as the reaction would take place at a constant PH throughout. In addition, PH is the independent variable I am testing; therefore, its constancy is important.
Variables in the experiment will need to be controlled and the following need to be kept constant
- Concentration of trypsin
- Amounts of reagents
- Enzyme to substrate ratio
- Temperature
- Method of measuring solutions
I will use equal amounts of trypsin, milk, PH, buffer and distilled water in all the experiments and will use the same measuring equipment to ensure fair representation of the effect of different PH on digestion. If variables aren’t adequately controlled then the data collected would be erroneous and would not represent the true effects of PH on digestion as altering variables could lead to changes in rate of reaction.
As we know the collision theory states that the more collisions in a system, the more likely the molecules will react and therefore the faster the reaction. Rates of reaction can be affected by variables such as concentration, as more molecules of the substance are present therefore increasing the chance of a collision, another is temperature.
All enzymes have an optimum temperature at which they work the fastest, this is because more kinetic energy is available and particles move faster resulting in more collisions and hence more enzyme- substrate complexes forming. Therefore the reactions will be carried out at 40oc as this is the optimum temperature for trypsin, using a higher temperature would cause hydrogen and ionic bonds in the tertiary structure of the enzyme to break causing them to denature. The temperature will be maintained using an electronic water bath as it would be more precise than one made using a beaker and Bunsen burner, as the temperature is easily maintained.
Controls will be set-up alongside the experiment to illustrate that the results obtained are due to the breakdown of casein by trypsin working in different PH and not due to the change in PH itself.
The solutions will initially be equilibrated so that they are all at the required temperature before the reaction begins, this way, part of the reaction won’t take place at a lower temperature whilst it rises to the required one, this will make it more accurate
The dependant variable is the amount of breakdown of casein by trypsin; I will measure this using colorimetry to calculate percentage transmission. Colorimetry is a measurement of the wavelength and intensity of electromagnetic radiation in the visible region of the spectrum. It is used to identify and determine concentrations of substances; a higher concentration of coloured solution absorbs more and transmits less light than a solution of lower concentration. The breakdown of Casein in milk causes it to become more translucent, this would suggest that the more the digestion, the higher the translucency, therefore using this method should prove to be suitable and a more accurate way of measuring digestion, rather than qualitatively analyzing the colour change.
The blank is prepared using HCl as it would cause the casein to be fully hydrolysed and would show optimum digestion and can then be used to calibrate the colorimeter.
Although the reagents would diffuse from a high concentration to a low and mix, this will take-up experimental time and may take different times for each, therefore shaking all the test-tubes will ensure that the reagents are mixed initially.
I intended to use immobilized enzymes as they would be easy to remove in order to stop the reaction i.e. Alginate beads which can be sieved. However we know that immobilization may lead to loss of enzymatic activity e.g. due to the immobilizing framework hindering the arrival of substrates into the active site. Therefore, when removing the test-tubes from the water bath and placing solutions in the colorimeter, I will have to work quickly as immobilized enzymes won’t be used and the reaction will not be stopped.
Finally repeating the experiment thirty times would allow better evaluation of the experiment and provide sufficient data to carry out statistical tests.
