My prediction graph will be able to the rate of reaction with increased concentration of lead ions
Inhibitions are likely to react differently with different types of enzyme so the rate of reaction of both enzymes would be different. One difference would be size of active site enzyme could lead to have faster rate of reaction in Fungal amylase compared to Bacterial amylase. Different results in the reaction of both enzymes would enable to find out the answer by looking at their protein structure. Bacteria and fungi due to different conditions they grow in they could have different resistance to inhibitors such as lead ions .
Control variables
Temperatures - This can affect the rate of reaction if done in cold environment that means that it will take longer for experiment to perform. It is also important that it not done near sunlight which provide heat which might help to increase the rate of reaction. This experiment has to be done in a shade. There could be heat sources from other parts of room so doing this experiment in air-conditioned space.
PH- this can alter the ionic charges in an enzyme changing the chemical structure of enzyme and losing its specifity. So its important to keep the PH environment same. With Amylase the appropriate PHwould be neither too acidic nor too alkaline. It is also important to use equal amount of buffers in all experiments. Buffers will eliminate little changes that there can be in PH.
Substrate and enzyme concentration- the amount of each bacterial and fungal Amylase and the concentration of starch used will be the same in each experiment. It is very important that when putting amount of enzymes and its substrate in I use very accurate measuring equipment because one drop of substrate or even a little mass of an enzyme is added in exceeded amounts this could affect the accuracy of experiments.
Time - The time for which experiment will be performed will be same amount of time. There will be certain human error, as we cannot the time accurately to hundredth of a second but in that little amount of time there are unlikely to be any change in rate of reaction. It is also important to know the time each enzyme is added to each test tube as enzymes put into one test tube might earlier might star the reaction more quicker but if the time differences are little then it should not have an effect on overall rate of reaction
Also it is very important to swirl the beaker because then enzymes would be evenly spread around the substrate solution and not concentrated at one particular area, which will slow the rate of reaction.
Apparatus required
Incubated flask – Doing this experiment in normal test tubes will create temperature problems. In order to measure the rate of enzyme effectively use incubated flask in which temperature of enzyme will be controlled in all experiments.
Buffers- In this experiment I will need a Citrate buffer. This is a mixture of citric acid and disodium phosphate in appropriate proportions to give the required PH. These two chemicals are able to absorb either added acid or added base by reacting and effectively neutralizing the environment. Buffers around PH 6.5 will be needed.
Dropper bottle of starch solution- we will place five droppers full of starch every experiment we do and all of these size of drops have to be equal.
Dropper bottle of lead chloride- this experiment is concerned with increased concentrations of lead ions so firstly I will begin by adding one drop of solution to both bacterial and fungal amylase
Pipette- this will be used to draw out the exact volume and starch of into incubated flasks. They are very accurate for measuring right volume of liquid to have fair rate of reaction.
Dropper Bottle of glucose solution- this would enable to see whether Glucose is being produced in the experiment.
Stop clock - I need to know what time I have added Enzymes into each test tubes. There is only a limited amount of time available for this experiment. As soon as enzyme is put into the test tubes I will start the stop clock and then after a certain amount of time I will measure the concentration of glucose formed.
Measuring the amount of products formed:
It is very important for me to analyse quantitatively the concentration of glucose. This why just measuring the colour change would not be suitable. We could use the clinistrix strips, which will change colour according to different concentrations of glucose. I want to measure the amount of product deposited by using a spectrophotometer Glucose in different concentration form different colours but in the ultraviolet range of spectrum. Since the amount of colour is directly proportional to the amount of starch digested you can make quantitative measurements of enzyme activity. In other words the more intense the blue is the higher the rate of enzyme activity.
I also need the reagent, which is required to work the spectrophotometer.
I will use the reagent Iodine, which turns dark black with the starch, not hydrolysed by amylase but as amylase will digest away starch the colour of starch would get lighter in colour. Then measure the absorbance by the spectrophotometer. The more lighter the colour Iodine gets the faster the rate of reaction.
In order to ensure good reading on spectrophotometer, Flask must be clean and dry from outside. There are should not be any bubbles floating around as we are not analysing air particles and good half amount of incubated flask should be filled.
I would need two incubated flasks in which one of them there would be bacterial Amylase and one in fungal Amylase.
Once the reaction is finished flask would be thoroughly washed and dried while proceeding with the next experiment.
I need at least 6 beakers of starch, glucose and lead chloride.
Each experiment would be allowed to proceed for 8 minutes at least. I will repeat the experiment, as many times are available for each experiment.
Wooden stir sticks would be used to stir the solutions well.
TEST FOR lead ions
- IN A CLEAN Incubated flask, place 0.5 ml of Bacterial Amylase into tube A. Add 1 drop of lead nitrate, stir and wait 30 seconds. Add half incubated full of starch (Be sure to stir the starch up). After 5 minutes add 1 dropper full of GLUCOSE TEST SOLUTION. Wait 5 minutes and record your results. After the test solution has been added, .
- What has the lead done to the activity of the amylase?
- Repeat the above procedure with fungal amylase for a particular concentration of lead ions.
- Add Buffers to eliminate little variations in PH.
Once the results are accumulated wash the equipments with distilled water and then start the next experiment with different concentration of lead ions on both enzymes.
Control experiment
In a control experiment it is important to measure the activity of amylase
- I will Place about 2 drops of starch in tube A of a incubated flask, and 2 droppers full of glucose solution in tube C. I will add 2 drops of the iodine solution to each. I will Record any colour changes. The colour observed in tube A is a standard for the presence of starch. I will be sure to stir up the starch solution each time before you add it to a tube!
- I will Place 1 drop of starch solution in depression B and 1 drop of glucose solution in D. I will then add 1 drop of glucose testing solution to each and compare colours. Record your results. Does starch contain any detectable sugar? Depression D is the standard for glucose.
- I will then Place about 0.5 ml of saliva in G, and then add 2 droppers full of starch solution. Let set for 10 - 15 minutes. Now add 1 dropper full of GLUCOSE TEST SOLUTION to G. I would then Record my results. What has the Amylase done to the starch solution? I will then record the results.
- Place 0.5 ml of saliva into depression F, add 4 drops of GLUCOSE TEST SOLUTION and note the colour change. Would the solution contain any glucose?
Risk assessments and hazards
Handling of both enzymes must be extreme care if there are to work properly in an experiment. There will be some toxic materials used in this experiment. Inhalation can cause breathing problems and cause damage to the body.
If enzymes are in powdered form then it can cause eye problem. Wear goggles to give protection to your eyes.
About harmful liquid solutions getting into your eyes, which can be very irritant so rinse your eyes immediately. Some solutions could also spill in your skin. If this happens rinse your skin immediately. You can reduce the risk by wearing disposable gloves.
It is important to maintain cleanliness because minor impurities may ruin a good bio-chemical experiment.
It is also very important to label different containers so we do not get the solutions mixed up. This involves less risk as it does not involve anything being heated up so less chance of skin burn.
Ethical implications
It would not be ethical to use a source of enzymes from animal cell sources.