The rate of the reaction without any external means of providing the activation energy continues at a much faster rate with an appropriate enzyme than without it. The maximum rate that any reaction can proceed at will depend, among other things, upon the number of enzyme molecules and therefore the number of active sites available.
Enzyme concentration
At low enzyme concentration there is great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate.
Eventually, increasing the enzyme concentration beyond a certain point has no effect because the substrate concentration becomes the limiting factor.
Preliminary:
Before the actual investigation I will have to carry out some preliminary research to see which method is most suitable for the investigation and what values I should give to constant variables such as substrate concentration (starch concentration in this case).
We are investigating the hydrolysis of starch. Starch is a polysaccharide made of repeating glucose units that are linked by α(1→4) bonds. Each repeating disaccharide (two sugar) unit is “α-amylose”. As well, there are branches in starch molecules caused by random formation of α(1→6) bonds. When starch is consumed, it is broken down into progressively smaller units, eventually becoming “maltose” (the two glucose molecule linked by the α(1→4) bond) and “glucose”. This hydrolysis of starch is brought about by the enzyme amylase.
Iodine ions (I5- and I3- ions in particular) fit very nicely into the core of the helical structure of the polysaccharide amylose found in starch, stabilizing the shape by making contacts with the sugar monomers. A blue color results from changes in the electron orbitals of the monomers and iodine ions when this occurs.
Iodine will also react with the amylopectin in the starch, but with a purple color rather than blue. This color change probably results from a similar interaction, modified by the branched nature of the amylopectin molecule.
All of the reactants in our case are colourless and accordingly, the effect of amylase concentration on the hydrolysis of starch can be found through colorimetric analysis by measuring the change in iodine staining for varying concentrations of amylase in the same reaction. There are two possible methods for doing so:
- Visual human judgment; taking samples from the reaction vessel and adding Iodine solution at intervals of time. The end of the reaction will be as soon as the experimenter observes a revert to natural Iodine colour (orangey brown) seeing that this will mark the absence of starch (fully digested by amylase)
- Using a Spectrophotometrer; as the intensity of the blue colour decreases, more light is transmitted through the solution. The amount of light absorbed can be measured accurately as absorbance using a spectrophotometre.
Each one of the two methods has its own set of advantages and disadvantages:
Visual human judgment:
Advantages – easy to conduct
Disadvantages – high chance of misjudgment
Spectrophotometer:
Advantages – very accurate, easy to use, quick analysis
Disadvantages – sensitive to noise (including photon and electronic noise), equipment too expensive, chance of poor instrument design
We do not as GCSE students, have access to spectrophotometers, therefore, our instructor suggested that the rough visual method would be the most appropriate in our case.
Safety Considerations:
Inhalation of amylase may cause irritation to the mucous membranes and the upper respiratory tract, so don’t get too close to the reaction vessel
Take care when handling amylase solution as it may cause skin irritation. In case of contact immediately flush area with plenty of water.
Eye protection must be worn, amylase may cause eye irritation.
Care should be taken using iodine, as contact with the skin can cause lesions; iodine vapour is intensely irritating to the eyes and mucous membranes.
Apparatus and Chemicals needed:
We will need the following materials for the experiment:
- 1% stock amylase solution
- 1% stock starch solution
- Water
- Iodine solution
- Three measuring cylinders
- Pipette
- Spotting tile(s)
- Stopwatch
Constant Variables:
Temperature: enzymes work best at an optimum temperature.
Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too.
Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work. In this case, the experiment is going to be carried out at room temperature (approximately 22°C). However, I am unable to accurately control the temperature of the environment.
pH: as with temperature, enzymes have an optimum pH. If the pH changes much from the optimum, the chemical nature of the amino acids can change.
This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured. In this case the pH will be kept neutral and I will be very careful to both wash out and dry the equipments so that the pH of the reactions will not be altered by the reaction products of previous users.
Substrate concentration: at a low substrate concentration there are many active sites that are not occupied. This means that the reaction rate is low.
When more substrate molecules are added, more enzyme-substrate complexes can be formed. As there are more active sites, and the rate of reaction increases.
Eventually, increasing the substrate concentration yet further will have no effect. The active sites will be saturated so no more enzyme-substrate complexes can be formed. I will only use the stock 1% starch solution from the same container, which is stored safely in the laboratory so the substrate concentration will be constant.
Presence of inhibitors: Inhibitors slow down the rate of a reaction.
Reversible inhibitors:
Competitive reversible inhibitors: these molecules have a similar structure to the actual substrate and so will bind temporarily with the active site. The rate of reaction will be closer to the maximum when there is more ‘real’ substrate, (e.g. arabinose competes with glucose for the active sites on glucose oxidase enzyme).
Non-competitive reversible inhibitors – these molecules are not necessarily anything like the substrate in shape. They bind with the enzyme, but not at the active site. This binding does change the shape of the enzyme though, so the reaction rate decreases.
Irreversible inhibitors – these molecules bind permanently with the enzyme molecule and so effectively reduce the enzyme concentration, thus limiting the rate of reaction, for example, cyanide irreversibly inhibits the enzyme cytochrome oxidase found in the electron transport chain used in respiration. If this cannot be used, death will occur. Only tap water is available in the laboratory which may have resulted in some inhibition due to the presence of fluoride.
Total Volume: the total volume should be constant so that we can compare the results for the different concentrations it is the 100% value in this experiment.
Independent Variable
Amylase concentration
Dependent Variable
The time taken for polysaccharide starch substrate to be fully digested into disaccharide maltose.
Implementation & Analysis
Procedure for experiment
- Collect the apparatus needed, wash and dry it accordingly to get rid of any contamination
- Using the Iodine dropper place a globule of Iodine in each concave on the spotting tile
- Measure the quantity of amylase, starch and water to the decided amount using three separately marked measuring cylinders A, S and W so you do not get mixed up between the chemicals; they all acquire the same physical state, appearance and odour.
- Dilute the amylase solution to the decided concentration by adding the water measured out beforehand
- Pour the starch into the dilute amylase solution and start the stopwatch instantly
- With a clean pipette, take a sample from the reaction vessel at each 30 seconds interval and add a drop to the Iodine in one of the concaves on the spotting tile
- The blue colour will eventually fade owing to the decreased starch level, a return to the standard orange Iodine colour will mark the end of the reaction, hence, the stopwatch should be stopped at this moment.
Preliminary Results:
- Deciding the values of the total volume and the amount of starch
- Deciding the highest values of water and amylase
Total volume = 60 cm3
These take too long; try a total volume of 40 cm3
1 minute 32 seconds – this is a comparatively realistic amount of time to be spent on an experiment
My decided values:
The highest value of amylase – 15 cm3
The highest value of water – 10 cm3
The total volume will be 30 cm3 instead of 40 cm3 to speed the reaction further due to limitations of time
Table of values:
Results:
1st trial:
2nd trial:
3rd trial:
Average:
The graph shows a negative gradient, m
y (time) decreases at a constant rate
it is proportional to –x (concentration) plus a constant
so according to the equation: y=mx+c (m is negative)
This complements the recognized theory of enzymatic reactions:
At low enzyme concentration there is great competition for the active sites and the rate of reaction is low (takes more time). As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate.
Conclusion & Evaluation
Precision of measurements & accuracy of experiment:
Experimental error is always with us; it is in the nature of scientific measurement that uncertainty is associated with every quantitative result. This may be due to inherent limitations in the measuring equipment, or of the measuring techniques, or perhaps the experience and skill of the experimenter. It goes without saying that, when conducting this experiment, I endeavoured to be as careful and as accurate as I could in my measurements. The method that was used is fairly accurate because the results all make sense. The observations that were made were accurate but there were still factors that were beyond control like the temperature and humidity of the air. The measuring cylinder is a fairly accurate measuring device for liquids such as amylase and starch because it is marked appropriately, and every care was taken to make sure that any human errors like misreading the markings, and measuring incorrectly were kept to a minimum. However, there are several areas where precision could be improved:
I had to measure out my amylase, starch and water using a measuring cylinder which was set up on one of the lab benches. I found this a little difficult to read at times, as they were placed significantly lower than eye level. Therefore, it was not always possible for me to measure out these volumes as accurately as I would have liked.
Measuring cylinders are also less accurate than other liquid volume measuring devices:
You can measure the volume of a liquid with a measuring cylinder, pipette or burette. Measuring cylinders are least accurate. This may not matter if another measurement is even less accurate, or if you want excess of a liquid. If the cylinder has graduations (marks) every 1 cm3 like ours, then when you measure for example the 10 cm3 of water you can be sure you have more than 9.5 cm3 but less than 10.5 cm3. In this case your error is ±0.5 cm3 in 10 cm3, and the percentage error is 0.5/10 x 100 = 5%.
If you had measured 50 cm3 with the same measuring cylinder the error would have been 0.5/50 x 100 = 1% so the bigger the reading the smaller the percentage error.
If you use a big measuring cylinder the graduations may be every 2 cm3. If you measure 50 cm3 with one of these you can be sure that you have more than 49 cm3 but less than 51 cm3, so your error would be 1 cm3 in your reading, I used the smaller ones hopefully!
Pipettes are more accurate than measuring cylinders. Most school pipettes are made to an accuracy of one drop when they are used correctly.The volume of one drop = 0.05 cm3. A 10 cm3 pipette has an error of ±0.05 cm3 in 10 cm3. The percentage error is 0.05/10 x 100 = 0.5%.
Burettes are also more accurate than measuring cylinders. They have graduations every 0.1 cm3, so when you take a reading it should not be more than 0.05 cm3 too high or too low. However, when you use a burette you take a reading at the start and the end , so you have two errors of 0.05 cm3 i.e. total error = 0.10 cm3.
Looking for an end point in the reaction can be very subjective. Although this reaction had a fairly definite end point, it would still be difficult to decide when the Iodine had retained its normal colour and starch was wholly absent. The human eye differentiates about 300 hues and 100-150 luminance variations. The best resolution is for green and red; less is for blue so this obviously has an impact on the experiment. A high quality spectrophotometer would standardize this aspect of the experiment by measuring the intensity of the blue colour; as the intensity of the blue colour decreases, more light is transmitted through the solution. A spectrophotometer measures the amount of light absorbed accurately.
The structure of a spectrophotometer:
I have tried to restrict all calculations to a sensible and meaningful number of significant figures, as a consequence of the possible errors and inaccuracies mentioned above.
Different kinds of error
Several kinds of errors are usually present in experimental data. Their effects on the desired results can range from insignificant to disastrous, depending on how well they are understood and accounted for.
Systematic errors are consistent effects which change the system under study or the measurements you make on it. They can consist of, for example:
- Uncalibrated instruments (balances, etc.)
- Impure reactants
- Leaks
- Temperature effects not accounted for
- Biases in using equipment (even numbers in reading scales, seeing hoped-for small effects, etc.)
Systematic error affects the accuracy of an experiment but not the precision. Repeated trials and statistical analysis are of no use in eliminating its effects. Careful experimental design and execution is the sole approach to reducing systematic error. In this particular experiment, systematic error could have come from the temperature of the environment, which I was unable to control. Indeed, I noticed that, as the experimental session continued, the atmosphere in the laboratory did seem to warm up. If they were available, I would have liked to use an electronic water bath to keep the temperature constant.
An electronic water bath:
The dual thermostats would provide optimum protection for my work and water bath. If the bath temperature would exceed my pre-set limit the high limit alarm would alert me.
A secondary Safety Set thermostat would guard against thermal runaway, automatically disconnecting heater power should bath temperature get too high or the liquid level drop too low.
I do not think that there were other factors that would have caused drastic systematic error in this case.
Random error arises from mechanical vibrations in the apparatus, electrical noise, uncertainty in reading of scale pointers, and other "fluctuations". It can be characterized, and sometimes reduced, by repeated (at least three) trials of an experiment. One set of measurements alone is not usually very reliable. In order to obtain reliable results, it is necessary to carry out the investigation more than once. Obviously, the more replicates, the more reliable my results will be. This however, has to be balanced against the time constraints of repeating the procedure numerous times. I only had time to take three sets of measurements. It was opportune, therefore, that my results contained no glaring anomalies. This was borne out by the resulting line graph. If I had had more time, however, I would have liked to have replicated the experiment at least twice more, and then averaged out the results, in order to minimise random errors and impart more credibility to them. (Note that random error affects the precision of an experiment and to a lesser extent its accuracy.)
As I have previously mentioned, I believe that the positioning of the measuring cylinder accounted for some error in measuring out the volumes of hydrochloric acid for this experiment.
Extension
This experiment could be extended to investigate the effect of another variable on enzyme activity. The effect of cigarette smoke on enzymes is an interesting topic:
Two effects of smoking that I know already are on the enzyme inhibitor a1-antitrypsin and on the enzyme elastase (a protease). a1-antitrypsin is secreted by lung tissues to prevent endogenous proteases digesting the proteins in the lung. Neutrophils (cells) in the alveoli of the lung release a protease called elastase. a1-antitrypsin inhibits elastase and other proteases by tightly binding to the protease.
Smoke affects this system in two ways. Firstly in response to the irritant nature of smoke, more neutrophils are attracted from the blood into the lung tissues, thereby increasing the local level of elastase activity. Secondly, oxidising agents in the smoke destroy the a1-antitrypsin, by turning an important -S group to S=O in a methionine residue of the protein. So the protease levels are increased and the ant--protease levels are decreased, resulting in proteolysis (autoproteolysis) of lung tissue, which we call emphysema.
Due to the high levels of oxidising agents (free radicals) in smoke, other enzymes that increase in activity are also the body's anti-oxidative protection systems, which include enzymes such as catalase, superoxide dismutase and glutathione peroxidase and glutathione reductase.
Conclusion
My initial aim was to investigate the hydrolysis of polysaccharide starch substrate into disaccharide maltose and find out how varying amylase concentrations affected the reaction time.
I successfully conducted the experiments that enabled me to do this. In addition, I confirmed my prediction that the time taken for the reaction is proportional to the rate of reaction; that is, the higher the enzyme concentration the less time is consumed.
As can be seen from my results tables, during the sixth run of the experiment, where the amylase concentration was at its highest, the starch substrate was hydrolysed more quickly (89.33 seconds). Conversely, in the first run of the experiment, where the amylase concentration was at its lowest, a long time was consumed before the starch was fully hydrolysed.
This indicates that the higher the concentration of an enzyme, the faster the enzymatic reaction will be for this experiment.
Bibliography:
Letts Revise AS & A2
S-cool! – AS & A2 Level Biology Revision – Quicklearn
www.enzyme.co.uk
