Fair test: In order to make sure that the test is fair I need to make sure that all variables remain constant except the one which I am investigating which is the temperature of the solutions. This is to ensure that any changes, which may occur to the amount of maltose produced, are due to the change in the temperature of the solutions and not any other external factors. I will ensure that the ph of the solutions remain the same by using buffer so a constant ph is maintained. I will stop each reaction after exactly 10 minutes, to make sure this done through out the solution I have to mix the solution after I have added the DNSA to ensure the reaction is stopped through out.
Safety: This is not a very dangerous experiment however I will have to be careful when handling the test tubes, as they may be hot.
Method: First I will place 1.0ml of 0.5% starch solution and 0.5ml of ph 6.5 buffer solutions in 11 separate test tubes and label 10 of them with the temperatures they will be heated to. The range of temperatures will be 0, 21 (room temp.), 35, 40, 45, 50,55, 60, 65 and70. The other test tube will be labelled “BLANK” and left t room temperature, ideally I would have a blank for all temperatures but however due to time constraints this will not be possible. I will then also add 0.5ml of distilled water to the “BLANK” only. The test tubes will then be placed in the appropriate water baths at the labelled temperatures and 0.5 ml of amylase solution with chloride will be added to each test tube in turn at one minute intervals except the BLANK to start the reaction. Adding 1.0ml of 3,5-dinitrosalicylic acid reagent into each test tube after each solution has been reacting for exactly 10 minutes will stop the reaction. I will make sure that the solutions are mixed well after the 3,5-dinitrosalicylic acid reagent has been added to ensure that the reaction has been stopped through out the solution. In order to develop the colour I will then remove the test tubes from the water baths and place them into a boiling water bath for ten minutes, when they are removed I will leave them to cool before adding 5.0ml of 0.15M sodium chloride to each tube to increase the volume of the solution and dilute the mixture. I place a bit of each test tube into individual curvettes before reading the absorbance value at 550nm in a colorimeter using my BLANK as the zero setting. I will try and repeat the experiment to get an average absorbance value for each temperature.
Preliminary experiment: I performed a preliminary experiment before starting this investigation; this tested the effect of different temperatures on pancreatic amylase, I performed tests at two different temperatures, one was at 50 C and the other was a blank at room temperature (21 C). The results were as follows:
The preliminary results are not that reliable a basis for my prediction because I only did two different temperatures and I needed a larger number of different temperatures to hep get a more reliable basis for my prediction.
Prediction: Based on my background knowledge and my results obtained from my preliminary experiment I predict that the higher the temperature is, the higher the rate of reaction of enzymes will be up until roughly 55 C which I believe is the optimum temperature. After the temperature rises higher than 55 C the rate of reaction will begin to fall as enzymes start to become denatured because the high temperature alters the shape of the active site meaning that substrates can not enter and react.
Quantitative prediction:
I will record my results using a table of results and a graph showing a line of best fit. There will be two sets of results from which I will find an average.
Sketch Graph:
Obtaining
Results Table: The effect of different temperatures on the activity of pancreatic amylase.
I WAS NOT ABLE TO DO ANY REPEATS DUE TO TIME CONSTRAINTS.
Analysis
Analysis of results: I can see from the results table and the curved graph that I obtained, that my prediction that “the higher the temperature is, the higher the rate of reaction of enzymes will be up until roughly 55 C which I believe is the optimum temperature. After the temperature rises higher than 55 C the rate of reaction will begin to fall as enzymes start to become denatured” was correct. However my quantitative prediction was not quite as accurate as I had hoped it would be. I predicted that the highest amount of absorption would be 1.2 but it turned out to be slightly lower at 1.15, the lowest was 0.00 as I had predicted. I found that there was a slow rise In absorbance up to 20 C but after that there was a sharp increase up until 50 C after this temperature the absorbance value began to fall extremely quickly and by 70 C the absorbance was 0.The reason for the low absorbance values between 0 C and 20 C is that at these temperatures not many enzymes have kinetic energy so there are fewer collisions and fewer reactants reach the activation energy necessary for a reaction. This results in fewer reactions. After 60 C the absorbance values fell rapidly, this is because at temperatures higher than that the shape of the active site is altered and so substrates cannot react with the enzymes. The average gradient of the graph was . The Q10 of the graph was found to be .
Sketch Graph:
Conclusion: I conclude from my results that temperature can have a large effect on the activity of pancreatic amylase. If the temperature is too low or if it is too high then there will be no activity at all and no reactions will take place because either the reactants do not have the required activation energy or because the shape of active sites of the enzymes are changed when the hydrogen bonds holding the secondary and tertiary structure of the enzyme together are broken. I believe that the optimum temperature for the amylase enzymes falls between 45 C and 57 C.
Evaluation
Improvements: In order to improve this investigation I could increase the number of different temperatures that I test so that I have a clearer idea of where the optimum temperature for this investigation lies. To improve the investigation it would also be useful if the water baths heated the test tubes to the temperature, which the display on the outside has. Using a newer colorimeter would make the results more reliable as the one I used was quite old and possibly slightly inaccurate.
Reliability and Accuracy: I believe that the results from this experiment are both reliable and accurate as nothing in my method would result in me having completely inaccurate results. I was very careful that all precautions to keep all other variables other than the temperature at a constant level, followed however when filling the colorimeter containers small amounts of the solution ran down the sides of the curvettes on occasion and also some of the curvettes had scratch marks on them, this may have resulted in slightly less accurate results but did not seem to have a mayor effect. I believe that my method was good as the results I obtained agreed with my theory. The results still followed the general trend of the model rate/temperature graph shown underneath my paragraph on background. It was also a fair and safe experiment as all precautions were followed regarding safety and only changing one variable.
Other possible experiments: There Other factors that I could investigate to further this investigation, for example I could test the effect of different concentrations of enzymes, the effects of different ph levels or the effects of different concentrations of substrate and the effect they would have on the activity of pancreatic amylase. I could also test to find out the different optimum temperatures and ph levels of different enzymes.
Conclusion: I believe that the investigation was suitably accurate and reliable and the conclusions drawn from the results to be correct however there were ways in which the investigation could have been improved and made more accurate. I am confident that my conclusion is accurate despite having to use an old colorimeter to obtain my results.
Bibliography
- Collins Gem, basic facts, Biology
- Collins advanced modular sciences: Biology As, Mike Bailey and Keith Hurst.
- CGP GCSE Double science Biology
