An Investigation Into the Effects of Temperature on Enzyme Action
An investigation into the effects of temperature on enzyme action An enzyme is a biological catalyst that speeds up the rate of reaction in certain biological functions.They play a vital role in many aspects of human physiology and are necessary for the functioning of a number of systems, for example in the digestive system to help to break down food.
All enzymes have a unique active site that can fit on to a particular molecular arrangement on a target substrate; a substance e.g.
carbohydrate, protein, or fat, that the enzyme is designed to breakdown.There are a number of different enzymes in the human body; each type produced specifically to perform a certain role. Enzymes are not themselves destroyed in the reaction to break down a substrate but their effectiveness is reduced under certain adverse environmental conditions. The two most important ones are temperature and PH level; also concentration of enzyme is also a determining factor. Enzyme action is biochemical in nature and, in line with many chemical reactions; it speeds up with an increase in temperature.
This would continue until a certain critical temperature is reached where it’s working efficiency drops suddenly.This is due to a deforming or ‘de-naturing’ of the active site leaving the enzyme unable to bond with its substrate. In humans this can have life threatening consequences if the body temperature rises above 40 degrees Celsius. As enzymes normally function in their own particular part of the anatomy, they operate most efficiently in a medium with a specific acid/alkali (PH) balance. If this balance is either too high or too low it’s functioning is reduced and in extreme circumstances this can lead to de-naturing also. An investigation was carried out in order to study the functioning of a particular enzyme ‘Trypsin’.This is produced in the pancreas and is found in the pancreatic juice; it is used to break down proteins.
As it is believed that all enzymes function most efficiently at some optimum (ideal) temperature this investigation looked at the ability of trypsin to function effectively over a range of discreet temperature settings. These included measurements from 20 degrees c, to 60 degrees c, in ten degree intervals and included a measurement at 0 degrees c, for comparison. The substrate used was protein from skimmed milk. Method A one per-cent solution of trypsin in sodium hydrogen carbonate was used to eliver a uniform dose in a favourable P. H. environment. A skimmed milk powder preparation of 5% in 95% purified water was used to mitigate undue discrepancy in the results.
All equipment, that came into contact with the study mediums, was sterile e. g. test tubes, syringes etc. A cross was marked with a marker pen, on the side of one test tube at the bottom and then 2ml of trypsin solution was then placed in a second. The two test tubes were then placed in one of a number of water baths, set at varying temperatures, for five minutes, in order to raise them (or lower) to a target temperature.The time was measured with a digital stop clock. After five minutes both samples were removed and the trypsin was poured into the tube containing the milk.
The stop clock was re-started and ran until the trypsin had broken down enough of the milk protein to make the marker pen cross clearly visible across the width of the test tube. This process was repeated, by five experiment groups, until each group had a measurement at each required temperature. The results were then recorded on a data table. All due health & safety precautions were followed including the wearing of lab coats and goggles.Care was taken when handling the samples at the upper temperature ranges e. g. ; 60 Celsius.
As trypsin itself is an irritant care was taken with this and hands were washed thoroughly. Nobody drank the milk. One or two checks and tests were put in place in order to validate the data trends. A colorimeter was used as a more scientifically consistent measure of milk clarity: (one sample was taken from each temperature setting) and mean readings were calculated from the table results to hopefully reduce the effect of moderate outliers.Conclusion As expected the results did indeed show an increase in trypsin activity as the temperature was raised above zero towards forty degrees Celsius. The only surprising result was that the data trends showed that this increase was sustained until a maximum was reached at 50 degrees c, when it was expected that the maximum would be at 40 degrees c, which is closest to its working temperature of around 37 degrees c. There are a couple of possible explanations for this apparent anomaly.
Firstly: Among the readings for the 40 degrees c, and 50 degrees c, samples there are a number of irregularities; including two low readings at 50 degrees c. It must be pointed out at this time that a shorter time recording indicates an increase in trypsin activity, so the table and graph are ones of inverse correlation; as the graph or data go down, the value (enzyme activity) goes up. Secondly: The samples were only placed in the water bath for five minutes and as soon as any samples were removed the temperature would have begun to gravitate towards room temperature.It stands to reason that the rate of change in samples that were furthest from room temperature, to start, would have been the greatest. Consequently, the sample that should have been 50 degrees c, to start, might have been considerably lower at the time of reaction. It is hard to believe that all of the trypsin would have been de-natured in one instant, so any that was not would have reacted rapidly causing a fast reaction time for the 50 degrees c, sample.Returning to the first point: As the trypsin reacted during the experiment, the marker pen cross would have appeared gradually as the milk sample cleared.
This would have left a large margin for error because the point at which to stop the timer would have been, very much, a matter of opinion. This has probably lead to a number of inconsistencies in the results. The effects on the data range seem to have been varied, for example: At 0 degrees Celsius, the difference between the highest and lowest recordings was 6%, at 60 degrees it was 241%.One check, previously mentioned, was the measurement of a data set using a colorimeter. This device measures the amount of light passing through a sample. The results from the back-up experiment confirmed the expected trends; namely as the temperature increased the trypsin activity increased up to an optimum of 40 degrees c, after which the enzyme activity tailed off rapidly. How the experiment could have been improved: Overall, the experiment was well conducted and good practice was followed.
However, having one person with experience in analyzing samples deciding exactly when to stop the timers, would have reduced the occurrence of any outliers in the data. Also, it is not difficult to imagine setting up equipment that would work along the lines of the colorimeter, measuring light levels through a sample, only set to stop a timer the moment a particular uniform reading was reached. The samples could then be placed in this equipment the moment the trypsin was added, hopefully resulting in an accurate and uniformly consistent set of data being extracted.