- Put 10ml of the substrate casein into the five remaining boiling tubes. Again label each boiling tube.
- Put all 10 boiling tubes into the water bath, for the temperature to acclimatize.
- Within the first minute, empty the casein into the first boiling tube. After every minute, repeat this step for the other 4 boiling tubes.
- Every 5 minutes take a sample out the boiling tube, using a pipette and fill up the cuvette.
- Taking care in not touching the ridged sides, place it into the colorimeter, to measure the transmission – how much light is passed from end to another (in %). This step in taking the reading from the colorimeter should take less than a minute.
- Put the sample back into the correct boiling tube of concentration.
- Record results.
- Access three more results from fellow peers, so an average can be taken.
Planning
The independent variable in this experiment is the different concentrations of trypsin. The dependent variable is the percentage transmission reading from the colorimeter. The controlled variable in this experiment is each boiling tube has the same amount of time (5 minutes) in the water bath. Also the measurements of all the solutions are going to be accurate and equal because a syringe is going to be used which is very precise.
As there are other groups also carrying out this experiment, after the experiments are completed, the results of two other groups are going to be collected. This is so an average can be taken and the results are more reliable. This way the data is not displaced by any anomalous results.
The apparatus and experimental procedure must be selected and used in a way that will ensure that the results are valid – results are valid if they measure what they are supposed to, and if they are precise, accurate and reliable. In this experiment, for the results to be valid, the concentration of the trypsin needs to be varied and the temperature needs to acclimatize before the experiment begins. The readings would not be valid if the concentrations were equal and the temperature was not acclimatized.
A faulty piece of equipment or incorrectly used apparatus may give precise readings but inaccurate results. In this experiment when using a colorimeter, using a single sample cuvette with dirt on the side will give precise readings, but they will not be accurate.
When experimenting, measuring instruments needs to be selected with an appropriate degree of accuracy. During this experiment, because I will need to measure volumes less than 1cm3, it is more suitable for a 1cm3 syringe to be used, rather than a 5cm3 or 10cm3 syringe.
The systematic errors could be when using the colorimeter, the cuvette could be dirty or the cuvette could be scratched, the error introduced would be the same throughout the investigation. The incorrect use of equipment could cause systematic errors. By not calibrating the colorimeter before use could cause errors. Additionally reading volumes in pipettes and syringes could cause faulty measurements (parallax on measuring). The water bath could be slightly out, because the temperature may not be exactly 300C.
The random errors may be reading the volumes, or not precisely measuring out the volumes. Carelessness, like not concentrating on times that is meant to use during the experiment.
Risk assessment
- Be careful not to bump into the water bath, this could spill the water, cause injury or cause people to slip and get injured.
- When transferring the liquids from different boiling tube, use tongs or heatproof mats to prevent scolding of the hands.
- Carelessness could cause the boiling tubes to break, causing an injury.
- Ensure all glass apparatus are kept in the middle of the worktop.
- Ensure goggles are worn because trypsin is a protease enzyme.
Results of enzyme concentration on the rate of reaction
Key
Trypsin concentrations
Minute(s)
Group 1 result for percentage transmission
Group 2 results for percentage transmission
Group 3 results for percentage transmission
Averages for percentage transmission
Analysis of results
The rate of an enzyme reaction depends on the concentrations of enzyme and substrate. As the concentration of the trypsin increases the percentage of transmission rate also increases.
Provided that the substrate concentration is high and that temperature is kept constant, the rate of reaction is proportional to the enzyme concentration.
The 1% trypsin worked best because the active sits of the enzyme molecules are still free and are able to be a site for the substrate to react. Whereas, with a lower concentration, there are less active sites to accommodate all the reactions, saturated.
The 0.00% line had no change and remained at 1%, because there was no trypsin solution added to, it was 10ml-distilled water.
In the first five minutes of the 0.25ml trypsin was at 13, which is a dramatic change from the 0.00% results. At 10 minutes it increased by another 10%, this result had a large error bar, ranging from 15 to 39. It then steadily increased again to 31, and then hastily again to 49. The steepest gradient on the 0.25% was between 15 and 20minutes.
The 0.50%, first result is 23, which is similar to the 2nd result on the 0.25% line. So already within the first 5 minutes, the time has already made a difference. It then increases at a fast rate and has a steep gradient, and reaches 54. After 54 it slowly increases to 56 and then 58. These results show that the substrate after 10 minutes is used up very quickly, so the rate of reaction is much slower.
The 0.75% line starts at 42 and increases another 10% after the first 10 minutes. The steepest gradient on the line is in between 5 and 10 minutes. After that is begins to level out, with the next result being 55 and then ends at 57, where the substrate has been taken up, that is the reason for the slight increase from 15 to 20 minutes.
The 1.00% starts at 50 and steadily increases to 54. The steepest gradient on the 1.00% line is between 10 and 15 minutes. At 20 minutes the percentage transmission is 62, so from the start of the experiment it has only increased by a total of 12. The gradient is a straight slant, which has almost leveled out; this is due to the enzyme molecules are saturated with substrate, so no further reactions can progress.
0.75% had the smallest error bars, whereas the to results which had the largest error bars were on 0.25% at 10 and 20 minutes. Though, the smallest error bar was also on the 0.75% line, which ranged from 30 to 32.
Errors
Calibrating the colorimeter could not have caused systematic errors, because the colorimeter was calibrated before use and checked. Reading and measuring out volumes incorrectly throughout the experiment could have caused errors. Even though time was vey specific and important in the experiment, there still could have been delays, which meant the solutions were left in the water bath longer then they should have been before taking a sample. The water bath could have been slightly out in the water bath.
Results were collected three times from each group so the mean from each concentration could be calculated, also this would improve validity of the results. To improve results each group could have carried out the experiment two or three times between themselves.
Evaluation
For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration up to a point, above which any further increase in substrate concentration produces no significant change in reaction rate. This is because the active sites of the enzyme molecules at any given moment are virtually saturated with substrate. The enzyme/substrate complex has to dissociate before the active sites are free to accommodate more substrate.
