Whilst carrying out this method I will be observing all standard lab safety rules (e.g. no running in lab). I will be wearing goggles at all times to ensure my eyes are protected from any hazardous substances, and I will using tongs to remove my tubes from the hot water baths to avoid risk of burning myself.
Enzymes are affected by certain factors, and to function at all they need these co-factors. To function well and efficiently enzymes need a suitable temperature and pH and there must be enough substrate available. Enzyme action can be stopped completely if inhibitors (such as sodium carbonate in this experiment) are present in the reaction mixture.
In this experiment there are two main variables I could change:
- Temperature the reaction takes place at
- The pH of the reaction
I will have to vary only one of these and make sure that I keep everything else constant to ensure a fair test.
Also to ensure a fair test I will make sure I keep all the amounts of the substances I use the same. I will be using a pipette and 1mm pipette tips for this, using these I can measure out very accurately the amounts of the solutions I am dealing with.
I think that I will do this experiment varying temperature, I decided on this because I know about how enzymes act at certain temperatures and I can therefore understand the investigation better. This means that I will keep the pH at a constant of pH5. I also think that I will record results at 5 different temperatures and repeat each temperature once. I will therefore use 5 different water baths, each at a different temperature, these will be 20°C, 30°C, 40°C, 50°C and 90°C. I also decided to do a control experiment to compare how the reaction would work without any enzyme in it. I also decided to use the following amounts of the substances in the reaction: 20µl of enzyme solution, 20µl of PPP substrate, 100µl of the buffer solution and 100µl of sodium carbonate.
For this experiment I am expecting to see that the enzyme flourishes at a temperature of around 40°C and therefore a lot of enzyme present at the end and a high magenta filter units will be recorded. Also I am expecting the amount of enzyme to be low when the reaction has been taking place at low temperatures (20°C) and at high temperatures (90°C). I am expecting a graph of my results with Magenta filter units on the Y-axis and temperature/pH on the X-axis to look roughly like this:
I think that this will happen because of what I know about enzymes. Heat supplies the reacting molecules with kinetic energy causing them to move faster. This means the chances of molecular collision are increased at higher temperatures, so it is more likely that enzyme substrate complexes will be formed. However, heat energy also increases the vibrations of the atoms which make up enzyme molecules. If these vibrations become too violent, chemical bonds in the enzyme break and the precise structure, essential for enzyme activity, is lost. Therefore at high temperatures enzymes become denatured. The ‘Lock and key’ theory is all about how an enzyme is precisely structured with a ‘lock’ in which a substrate (the ‘key’) can attach and bond. And so if the lock is damaged and misshapen the key can no longer fit and bond and will not form an enzyme substrate. This I what I expect to happen at the higher temperatures I am using. The lock refers to the enzymes “active site” The active site of an enzyme is the region that binds the substrate and contributes the amino acid residues that directly participate in the making and breaking of chemical bonds. The amino acid residues are called the catalytic groups. Enzymes differ widely in structure, function & mode of catalysis so active sites vary, but possible to make some generalizations.
Before I carried out my real experiment I did some preliminary work to familiarise myself with all the apparatus and the method. I got these results:
I thought that these results were not at all accurate or correct, I eventually decided that I hadn’t produced enough enzyme to put in the reaction. I then extracted some more enzyme, using more mung beans to get more enzyme out.
I was confident that this would then work, and started with my real experiment.
Observations
Observing all the basic lab safety rules (e.g. wearing safety glasses at al time) I carried out the experiment using the method I described previously and obtained the following evidence.
After obtaining and recording the results I can work out the averages for each temperature which will be easier to plot on a graph later.
Analysis
From simply looking at these results I can see that more product is produced going up to 40°C where the most is produced. I can also see that there is little product produced at the higher temperatures probably because most of the enzymes have been denatured.
I can produce a graph, using the average results to help show what the results are showing.
This graph with a best fit smooth curve shows that the enzyme action increases with temperature up to 40°C where it then begins to decline.
At 20°C the enzyme is not moving around very fast as the temperature is not high enough, this means little is formed at the end of the reaction.
At 30°C the enzyme is working faster than at 20°C and there is more product at the end of the reaction.
At 40°C the enzyme is working at its optimum temperature and here there is the most product at the end of the reaction.
At 50°C the enzyme has shown signs of it denaturing and some of the enzyme has been misshapen so not much is found at the end of the reaction. Denaturing is where the enzymes’ active site is mutated due to the high temperature; it results in the substrate being unable to now enter the active site so cannot be broken down into the product.
At 90°C you can clearly see there is little enzyme, this is because most of the enzyme has been deformed and so the substrate cannot combine with it, this is denaturisation.
This experiment showed that the enzyme could only sufficiently work at a temperature of about 40°C, this is its optimum temperature. At the temperatures below this the enzyme did not work as well, and at the higher temperatures the enzyme was denatured by the heat.
This supports prediction I made earlier based on my knowledge of enzyme action. I found that the enzyme did have an optimum temperature at which it worked the best and that the enzyme did denature at higher temperatures, which is what I thought may have happened.
Evaluation
I think that the method I used was sufficient for this investigation as it gave me more or less the results I would have expected.
The evidence I obtained was quite good, I got the results I pretty much expected, even if they were only roughly what I expected as I didn’t get a perfectly smooth curve on my graph.
I don’t think there were any real anomalies in the experiment, although my original preliminary results were certainly anomalous, and I but that down to a lack of enzyme solution in the reaction.
The suitability of the procedure was good, I don’t think that with the apparatus and time allowed I could have obtained much better results. The only possible thing that could have been improved was when the enzyme solution was being added to all the tubes individually before they were put in the water bath. The time taken for each tube to be filled and closed ranged, so that 1 pf the reactions would have already started whilst all the other tubes were being filled, this could have caused inaccuracy as each tube had a slightly different amount of time in the water bath. Possibly a multiple syringe so that all the tubes could be filled at the same time would help this problem.
I think the evidence is reliable enough and sufficient to support my conclusion. I think that my repeat results were a reasonable amount of time from my first results, had they been too far away the results could have been affected.
I think that to further this investigation I could record reactions which happen at 5°C rather than at 10°C intervals. This would mean I could find exactly what the optimum temperature is and also at what temperature exactly the enzymes denature.
I could also do this investigation using a different variable, such as pH. I could then see how this affected the action of the enzyme.
