Scientific Knowledge
Enzymes are proteins that serve as catalysts and accelerate chemical reactions in living organisms. Enzymes accomplish this by lowering the activation energy of the organism it is acting upon, however enzymes will only lower the activation energy of activation for specific organisms, reducing chaotic chemical reactions. The reaction is carried out in the active site of the enzyme. The substrate (organism acted upon), binds itself into the active site of the enzyme, and the chemical process begins. (4)
The enzyme’s active site will reform itself for the substrate in order to form a perfect bond, this is called Induced Fit. Another model of how enzyme works is called lock-and-key. It suggests that the substrate molecule fits perfectly in the enzyme’s active site like a lock and key.
There are numerous factors that can affect the rate of enzyme reaction, four have the greatest effects. They are the enzyme concentration, substrate concentration, temperature and pH. (5)
Sucrose is actually two sugars joined together (a disaccharide). Sucrose consists of a glucose and fructose molecule joined together.
Invertase splits the bond between the two sugars by hydrolysis to form glucose and fructose. Invertase belongs to a class of enzymes known as glycocidases. (7)
Enzymes react distinctively to alteration in the concentration of reacting molecules. At very low substrate concentration, collisions between enzyme and substrate molecules are infrequent(as there are less substrate molecules) and reaction proceeds slowly. As the substrate concentration increases, the reaction rate initially increases proportionally as collisions between enzyme molecules and substrate molecules become more frequent, because there are more substrate molecules present and therefore more enzyme-substrate complexes will form. When the enzyme begins to approach the maximum rate at which they can combine with reactants and release products, the effect of increasing substrate concentration diminish. At this point, further increase in concentration has no effect on the reaction rate. (2)
This is because at high substrate concentrations the active sites of the enzyme molecules at any given moment are virtually saturated with substrate. Thus any extra substrate has to wait until the enzyme/substrate complex has dissociated into products and free enzyme before it may itself complex with enzyme. Therefore at high substrate levels, both enzyme concentration, and the time it takes for dissociation of the enzyme/substrate molecule, limit the rate of the reaction (4)
My conclusion matched my prediction as the results, which I obtained show that the rate of reaction increases with an increase of the sucrose concentration from 0.5% to 4% and that when it reaches 8% concentration a maximum rate of reaction is reached and there is no increase after that (this can be seen on my rate of reaction graph).
Evaluating Evidence
The method used gives us the main steps for carrying out the experiment. It controls for other variables like the volume and temperature, as it tells us to use exact volumes of sucrose and invertase and also tells us to use water bath to keep the temperature constant. But it also gives limited details of the equipment that needs to be used and also lacks explanation of the procedure for the serial dilution of sucrose.
Other limitations of the method are:
- The method used doesn’t say what to use for the dilution of the 8% sucrose and also how much of that liquid is needed.
- The method doesn’t suggest how to get the water bath to 40*C and also how to maintain that temperature constant.
- The equipment needed to measure the time and the sucrose solution is not mentioned in the method.
- The method does not exactly say, when to place the clinistix in the sucrose solution.
- The method used doesn’t tell us how many of the sucrose filled test tubes to test at a time, and also how to time the time taken for the clinistix to turn blue.
- The method does not suggest how to make sure that the colour level of each clinistix is the same.
- Also the method does not mention washing up of the equipment whilst using it and does not account for safety precautions
What improvements would I make to the method or equipment
Used?
If I were asked to repeat the experiment I would change the method and equipment in the following ways:
- I would include a detailed list of the equipment needed. For example I would say that 2 cm3 pipette is needed to measure the sucrose solution, and also I would say that I would use a stop clock to measure the time taken for a colour change in the clinistix to occur.
- I would say that I would use a distilled water to prepare the different sucrose concentrations, by mixing it to equal amount of sucrose. I would also explain that the distilled water has a constant pH and this is one of the controls ensuring that the change of the reaction rate is only due to the Independent Variable (IV), which is the sucrose concentration.
- In my method I would include new equipment. One example is an electronic water bath. I would use it in order to maintain the desired temperature of 40*C constant, which would help for the accuracy of my results.
- I would also add more instructions of how to prepare the desired concentrations.
- I would also say that clinistix must be placed in the test tubes just before pipe ting the invertase
- To make sure that the colour of the clinistix is the same I would take one for control and compare it to the others by placing a white sheet of paper behind the samples so it’s easier to see the colour of each.
- And finally, I would add some safety precautions like eye protection and lab coats.
Were my results accurate?
I feel my experiment was a good procedure to use because it gave relative good results that were similar to what I had expected. However the method used lacked some details of how to carry out the serial dilution, and the equipment used was not the best in controlling the variables other than the independent. Therefore there were sources of errors including human errors.
Main sources of errors
- Carrying out the procedure for serial dilution might have resulted in incorrect dosage of the liquids needed for the dilution, giving sucrose concentrations other than the desired ones.
- It is difficult to measure out small amounts of liquids, in a continuous process, and thus the volumes of the different concentrations in each test tube might have been slightly different, resulting in non-controlled conditions for the enzyme to carry out it’s functions.
- The pipette used for the sucrose might not have been washed out after it had been used for pipetting sucrose and when used for pipetting invertase, it might have initiated a reaction between invertase and sucrose left in the pipette, thus affecting the time taken for the clinistix to turn blue.
- Keeping the sucrose concentration at a temperature of 40*C was very difficult without using an electronic water bath. I tried to control for other variables like temperature, by wrapping the beaker I used for the water bath in aluminium foil, and also by using tap water which was at 40-41*C. I used the foil in order to try to keep the water temperature constant. But I found it very hard to do that even though I constantly changed the water. Using this method of controlling the temperature I found rather difficult, as the temperature of the water was rapidly falling down and using tap water was not sufficient enough to maintain constant temperature of 40*C. Thus my results might not be as reliable as they should be in perfectly controlled conditions.
- It was difficult to press the start button of the stop clock at the exact time at which the pipetting of invertase is completed. This might have been different with each test tube resulting in the time, not been taken accurately which affects the results.
- There were two types of clinistix and they had different pink pads(one type had light pink pad and the other had dark pink pad). So if samples of both types of clinistix were used at the same time, this might have caused doubt in the colour level.
- Although I got approximately the same level of blue colour in each sample, it was still difficult to say when exactly that colour level was visible and when to stop the clock.
All these sources of error have influenced the accuracy of my results, but in the conditions I carried out this experiment they are almost unavoidable. As we are not using all the appropriate equipment there are some errors, but overall they are not significant and my results seem reasonable apart of one anomalous result on my second set of results at sucrose concentration of 8%. I consider this result that is 280 seconds to be an anomalous because it doesn’t fit in the general pattern which is that, the time taken for the clinistix to turn blue is decreasing with an increase of sucrose concentration. This result doesn’t fit with this trend because it is even higher than the time taken for the clinistix to turn blue, at the weakest concentration of 0.5%. This is impossible because even if a maximum rate of reaction is reached at 8%, the rate cannot decrease, but it can only stay the same at this concentration. Therefore the time taken for a reaction to take place at 8% cannot be higher than the time taken at 0.5%. This anomalous result I spotted immediately after I recorded it and in order to deal with it I decided to test the time taken to turn the clinstix blue, at concentration of 8%, two more times, in order to find an alternative result which fits in the general pattern. To substitute the anomalous result I decided to take the average of the two additional tests and disregard the anomalous. I will use my new result further on in the analysing evidence.
The results of the two additional tests were identical (162 sec), giving the same number as an average (162sec), which I believe to be a consistent result.
Now by looking at my results I see that at 4% from the same trial the time taken (160sec) is less than the time taken at 8% (162sec). This is not following the main pattern, but it doesn’t affect the average results, which are still showing the general trend.
The reason for the anomalous result at concentration of 8% might be because of many things. But the main reason for this result is that I wasn’t sure if the clinistix had reached the same level of colour, like the other clinistix, so I decided to leave it in for longer, and this is where I made a mistake because the colour didn’t change more than it was before. The reason for my uncertainty might have been because I’ve used different clinistix from the ones I used before.
The result at 4 % I don’t consider as a big anomaly, as it did not affect the average results.
References:
- Janet L. Hopson and Norman K. Wessells (1990)
Essentials of Biology- International Edition
- Ann Fullick (1994), Biology (Heinemann Advanced Science).
Heinemann Educational Publishers (Oxford) Ltd.
- N.P.O Green et al, (1990) Biological Science-Organisms, Energy and Environment,
Second edition. Published by the Press Syndicate of the University of
Cambridge.