Egg white or egg albumen is a globular, soluble protein, which trypsin will readily break down into its constituent polypeptides. As trypsin is an endopeptidase it will break the polypeptide chain into much smaller pieces. This has the effect of reducing the opacity of the egg white.
It is the effect that pH has on the action of the enzyme trypsin that is being studied in this investigation. At pH 6.5 – 7.5 both carboxyl and amino groups of amino acids are charged. Some amino acids have R groups that contain occurs where the enzyme will lose its tertiary structure and change the shape of the active site so that the substrate will no longer fit and fewer enzyme-substrate complexes form. pHs in-between may lead to partial denaturation. Changing the pH therefore alters the proportion of H+ ions, and hence will interfere with the electrovalent bonds and affect the three-dimensional or tertiary shape of the enzyme.
Changing the pH will alter the ability of the enzyme to cleave the polypeptide chain. Thus the effect of pH can be studied by observing the opacity of the egg white at differing pH using a colorimeter. At an optimum pH, there will be the greatest clearing of the ‘cloudy’ egg white. This can be confirmed by taking absorbancy readings using a colorimeter, the lower the reading, the clearer the mixture.
Pilot Studies
A pilot experiment should be conducted beforehand, to work out the best amounts of substances to use, and the duration of the experiment, to produce reliable results. Initially the range of pHs to be used should be tested, try 5 different pHs and see which produce the fastest rate of reaction, this should give a good idea as to the range of pHs that the enzyme will be active in.
At the best pilot pH differing volumes of Trypsin and albumen solution should be tested, to ensure that the final results give good quantitative data. For example, if too much enzyme is used the reaction will proceed too quickly.
The colorimeter should be tested and used to confirm that absorbancy readings decrease with the progressing reaction. The correct coloured filter should be determined it needs to absorb in the parts of the spectrum opposite to those that the solution absorbs, so it only lets through that wavelength light. Blue is probably the best as the egg albumen with the enzyme will be a yellowish tint.
Apparatus
( x 3 for three replicates):
14 test tubes
14 cuvettes
trypsin solution
boiled trypsin solution
distilled water
albumen / egg white solution
buffer solutions pH 1 - 12
water bath – 40oC
1 cm 3 and 2 cm 3 pipette and pipette fillers
colorimeter, filter, reference cuvette
marker pen
glass rod
Risk Assessment
A lab coat and goggles should be worn at all times during the experiment as they will minimise any substance contact with the skin or eyes in the event of a spillage. All enzymes, are potential allergens and have especially potent effects if inhaled as a dust, thus the trypsin will be kept in aqueous form and will be handled wearing gloves at all times.
Glassware should be handled carefully at all times and test tubes should not be raised above shoulder level. Special care should be taken with the electronic colourimeter, which to prevent electric shock should not be operated near any water source (water bath, sink) or with wet hands. When placing the cuvettes special care should be taken to prevent spillage.
Method
I will be using pH buffer solutions of pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
Increased temperature increases kinetic energy, molecules will collide more often and more molecules have sufficient energy to overcome activation energies, thus increased temperature will alter the rate of reaction. In order to ensure an accurate experiment temperature must be thermostatically controlled using a water bath set at 40oC.
I will also use control solutions, which are important in ruling out other extraneous factors as the cause of a change in the rate of the reaction. One control = boiled enzyme and albumen. The second control = distilled water and albumen. The controls show that the albumen is not digested without the action of trypsin.
The colorimeter should be set up with a coloured filter and set to zero using a reference cuvette containing a solution of albumen solution completely digested by trypsin.
Three replicates of each pH will be carried out to increase the reliability or results. Replicates also help to point out any anomalous results. 1 cm 3 and 2 cm 3 pipettes will be used for accuracy in measuring volumes of enzyme, buffer and substrate solutions. A stop watch, accurate to +- 0.01sec will be used to time.
1. Set up 2 sets of 14 test tubes and 1 set of 14 cuvettes; labelled from 1 through 13. Ensure that the test tubes are clean and that the cuvettes are free from dirt or grease that may interfere with the colorimeter reading.
2. Allow buffer, enzyme solution and egg white (albumen) solutions to equilibrate to 40oC separately in the water bath for ten minutes, then prepare the buffered enzyme solutions.
3. Add 1 cm 3 of the correct buffered enzyme solution, pH 1 to 12 to the correct test tube, held in the water bath. The control test tube should have 1 cm 3 of boiled enzyme in pH 7 buffer added.
4. Add 3 cm 3 egg white to the tube and as quickly as possible mix the solution in the tube by using a clean class rod.
5. Pour the solution into the cuvette up to the pre-indicated fill mark (4 cm 3 ) and place it into the colorimeter sample chamber with the clear sides in line with the light path, making sure not to touch the clear sides and in doing so leave fingerprints that may interfere with the reading.
6. Obtain an absorbancy reading for time 0, remove the cuvette from the colorimeter and place back into a holder in the water bath.
7. Record the changes in colour detected by the instrument at 30 second intervals for 10 minutes.
8. Repeat for each pH and the two controls.
9. Tabulate results and then plot a graph of absorbance units against time (minutes) for each pH. At the optimum pH, to start with, the absorbance reading should decrease rapidly as the rate of enzyme action should be high and then the decrease will begin to slow as most of the substrate (albumen) is used up in the reaction. During the linear portion of the curve, calculate the initial rate of reaction for each pH by finding the gradient: take a tangent and divide the y (absorbance) value by the x (time) value.
- By plotting the values for the rates of reactions against the corresponding pH the graph you produce should resemble a bell shaped curve, with the maximum / peak rate at the enzyme’s optimum pH. For most enzymes this is about pH 7-8 as it is the normal pH within the cell.
