Investigating the Effect of Copper Sulphate on Amylase Activity

Investigating the Effect of Copper Sulphate on Amylase Activity
The aim of my experiment is to observe the affect on amylase when adding copper sulphate to a starch solution. Introduction Enzymes are that act as catalysts, in other words they increase the rate of chemical reactions. Consider the following general reaction between two substances, A and B, which react together to form a product, substance C: A + B = C In biological systems, this reaction might occur very slowly, or not at all, in the absence of an enzyme. Enzymes will greatly increase the rate of formation of the product. They can increase the rate of reactions by a factor of at least one million. Most enzymes are large protein molecules, with complex three-dimensional shapes. Enzymes increase the rates of reactions by reducing the free energy of activation, so that the barrier to a reaction occurring is lower in the presence of an enzyme. The combination of enzyme and substrate creates a new energy profile, for the reaction, with a lower free energy of activation. Once the products have been formed, they leave the active site of the enzyme, which is left free to combine with a new substrate molecule. Enzymes, like chemical catalysts, are not used up in the reaction they catalyse so they can be used over and over again.
The overall reaction between an enzyme and its substrate can be represented by the following equation: ENZYME+SUBSTRATE à enzyme-substrate complex àENZYME + products Amylase is an enzyme that is found in saliva and pancreatic juice and aids the digestion of starch and other polysaccharides, which it breaks down into glucose, maltose and dextrins. It is required to digest carbohydrates (polysaccharides) into smaller units (disaccharides), and eventually converting them into even smaller units (monosaccharides) such as glucose. So Starch being a carbohydrate, Amylase hydrolyses the Starch. Inhibitors are substances that reduce the activity of enzymes. They act by interfering with the activity of the active site, either directly or indirectly. There are several types of inhibitors, which can be either reversible or (if they bind reversibly with the enzyme) or irreversible (if they bind permanently to the enzyme). Copper sulphate is known to be an Irreversible inhibitor. Irreversible inhibitors attach tightly to the enzyme molecule so that it loses its catalytic properties. Irreversibly inhibitors include ions of heavy metals, such as mercury and certain nerve gases. Theories that will help explain my reasoning Lock and key theory The basic mechanism by which enzymes catalyze chemical reactions begins with the binding of the substrate (or substrates) to the active site on the enzyme. The active site is the specific region of the enzyme which combines with the substrate. The binding of the substrate to the enzyme causes changes in the distribution of electrons in the chemical bonds of the substrate and ultimately causes the reactions that lead to the formation of products. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle. Substrate (key) The active site has a unique geometric shape that is complementary to the geometric shape of a substrate molecule, similar to the fit of puzzle pieces. This means that enzymes specifically react with only one or a very few similar compounds. This diagram will aid your understanding: [image] [image] [image] [image] [image] Smaller keys, larger keys, or incorrectly positioned teeth on keys (incorrectly shaped or sized substrate molecules) do not fit into the lock (enzyme). Only the correctly shaped key opens a particular lock. This is illustrated in graphic on the left. The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy. In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme). An enzyme inhibitor is a substance that slows down the rate at which an enzyme-catalysed reaction take place. Copper Sulphate is a non-competitive inhibitor which means that the copper sulphate molecules bind to a part of the enzymes tertiary structure disfiguring the enzyme and most importantly disfiguring the active site. Therefore the substrate is now unable to fit into the active site of the enzyme. Thererfore there will be a less concentration of working enzymes that could catalyse the reaction. Collision theory Theory that explains how chemical reactions take place and why rates of reaction alter. For a reaction to occur the reactant particles must collide. Only a certain fraction of the total collisions cause chemical change; these are called successful collisions. The successful collisions have sufficient energy (activation energy) at the moment of impact to break the existing bonds and form new bonds, resulting in the products of the reaction. Increasing the concentration of the reactants and raising the temperature bring about more collisions and therefore more successful collisions, increasing the rate of reaction. As copper sulphate is a non-competitive inhibitor it binds to the enzymes tertiary structure breaking peptide bonds as enzymes are proteins therefore disfiguring the enzyme and its active site. Less successful collisions will occur as there is a lower concentration of working enzymes. Therefore slowing down the rate of reaction. [image] The Variables My experiment has range of different variables, here are a few and a brief description of how they are going to be kept constant: Concentration of copper sulphate:- I will make sure I use 1% copper sulphate by reading the bottle. Concentration of amylase:- I will make sure I use 1% copper sulphate by reading the bottle. Volume of amylase:- I will measure out the chosen volumes of Amylase carefully. Volume of copper Sulphate:- I will measure out the chosen volumes of copper sulphate carefully. Temperature:- Temperature will be kept constant by using an electronic water bath. Ph:- We cannot use a ph buffer solution as it may be interfere with our experiment making our results unreliable. Pressure:- we are going to maintain the normal atmospheric pressure of 1. My dependant variable is the concentration of copper sulphate. My independent variable is the time taken to reach achromatic point. Hypothesis == I predict that copper sulphate will inhibit amylase activity. Reasons -?? I predict this because copper is known to inhibit the activity in Zinc in alcohol dehydrogenase and because amylase contains calcium which is also a metal, so it is reasonable to say that the copper will displace the calcium slowing down amylase activity. I also think that the copper sulphate will inhibit the amylase according to the lock and key and collision theorys. The lock and key theory tells us that enzymes break up bonds in a substrate by using an active site, the substrate being the starch in the food and the substrate has to have the shape to fit into the active site. The collision theory tells us that in order for something to react or to break a bond there has to be a collision of 2 particles, in this case it would be the enzyme and the starch. So when copper sulphate is added the CuSO4, which has almost the same shape as starch, the active sites are taken up by the CuSO4 particles leaving the amylase 1 less active site to use until the copper sulphate is released therefore the amylase having less successful collisions, therefore slowing down the rate of activity. Another factor to add to my reasons is this, Enzymes contain several amino acids with negatively charged residues. Copper, with it?s positive charge, would like to bind to these negative charges if it can gain access to them. That can upset the catalytic properties of an enzyme in a number of ways. For example, if the negatively charged residue participates in the chemical mechanism of catalysis, then having Cu bound is going to interfere with activity. This effect should be reversible. Cu can also have an effect at sites more distant from the active site of the enzyme if it disrupts the tertiary structure (the folded structure of the protein) of the enzyme. Again, these effects are most likely to be reversible, unless the Cu leads to unfolding of the protein. Very often, proteins cannot refold properly when they are unfolded. Equipment Test Tubes:- test tubes are needed to carry the substrate, the copper sulphate and the amylase, they are also used to mix the solutes. Thermometer:- Thermometers are used to measure the temperature of the solutes so it could b analysed as a fair test. Water bath:- Water baths are used to increase the temperature to a specific degree, so as it could not be a variable. Stop watch:- the stop watch will be used to time the intervals in which a sample of the mix should be taken out and tested with iodine. Pippette fillers:- will be used to fill up the graduated pipettes. Pasteur pipettes:- will be used to take out samples of the mixed solution to be tested. Graduated Pipettes:- These will be needed to accurately extract the chemicals and place them in the glass wear. I als selected the graduated pipette to use in my experiment due its low tolerance level of +0.06ml giving a tolerance level of 2 decimal places and percentage error of 0.24%. Iodine:- will be used to test the solution. 1% Copper Sulphate:- the substance we are testing if it is an inhibitor or not. 1% Stach Solution:- this is the substance we are using to test amylase activity after adding copper sulphate. 1% Amylase:- amylase Is used to break down the starch. White Dimple trays:- These will be used to add the iodine then the mixed solution to check if the starch has been broken down or not. Beakers:- These are needed for any dilutions that will have to be made. Pilot study For my pilot study I shall conduct a test to see what concentrations and volumes should be used to conduct a reasonable experiment. In light of the results of this experiment I will change the amount of concentration accordingly. To judge I would see what changes occur during the experiment, for instance, if the experiment takes too long I would have to decrease the concentration of copper sulphate. I would add different volumes of copper sulphate into the solution of amylase and starch and time how long it takes for me to see a change. I had to change the volume and concentrations of copper sulphate a number of times before I had a concentration/volume that was reasonable to use in my experiment. The 1st time I tested what concentrations I should use, I started with 0.1% copper sulphate with volume of 5cm3 and the temperature at 350. The result was inconclusive because my limiting factor was 12 minutes meaning any result after 12 minutes I would regard it as an inconclusive result. Inconclusive means that there was no result as my limiting factor was 12 minutes. concentration volume temp Time to reach achromatic point 0.1% Copper sulphate 5 cm3 35oC inconclusive 0.5% Amylase 5 cm3 1% Starch 5 cm3 The 2nd time I tested what concentrations to use, I used 0.01% copper sulphate with the volume of 5cm3 and the temperature of 350. concentration volume temp Time to reach achromatic point 0.01% Copper sulphate 5 cm3 35oC inconclusive 0.5% Amylase 5 cm3 1% Starch 5 cm3 The result was inconclusive so I conducted another test, with the concentration of 0.01% copper sulphate, the volume of copper sulphate as 2.5cm3, and the temperature as 400C. The result was that the Iodine didn?t detect any starch at the time of 500-520 seconds. Therefore I used these concentrations/volumes/temperature:- concentration volume temp Time to reach achromatic point 0.01% Copper sulphate 2.5 cm3 40oC 500-520 seconds 0.5% Amylase 5 cm3 1% Starch 5 cm3 Method 1. Prepare 4 different concentrations of the copper sulphate solution:
0.01
0.02
0.03
0.04
Volume of copper sulphate solution/cm 3_ Volume of distilled water/cm3 Final copper sulphate concentration 1 99 0.01 2 98 0.02 3 97 0.03 4 96 0.04 2. Set up a water bath at 40 0C 3. Pipette 2.5 cm3 of the diluted copper sulphate solution into one test tube and 5cm3 of starch into another test tube, Pipette 5 cm3 of amylase also and stand all three test tubes in the water bath and leave for several minutes to reach the temperature of the water bath. 4. Mix the copper sulphate and starch solutions together, replace the mixture in the water bath. Then add the Amylase to the solution and immediately start the stop watch. 5. At intervals of 20 seconds, remove a drop of the mixture and test it with iodine solution on a white dimple tray. 6. Continue the experiment until the mixture fails to give a blue-black colour with iodine solution. Record this as the achromatic point. 7. Repeat this procedure with the other concentrations of amylase. Use exactly 2.5cm3 of copper sulphate solution, 5cm3 of starch solution 5cm3 of amylase each time. 8. Repeat the whole process three times to gather averages. Risk assessment -?????? In my risk assessment I will be explaining how to make my experiment safe for me and the people that are around me. For example, Im going to use reasonable time intervals. This will enable me to handle my glassware with more care, for instance if the time intervals were very short there is more chance of either doing something wrong or dropping the glassware. I will make sure I am wearing goggles while I conduct this experiment so no harmful substances such could go onto the eyes. This is especially important in this experiment because I will b using enzymes which could digest the lining of the eye. Enzyme name Hazards Precautions to reduce risks Emergency procedures Storage and disposal Bacterial amylase All enzymes are potential allergies therefore should avoid contact and inhalation because it can cause asthma or irritate the membranes of the eyes and nose. bacterial enzyme in its powder form is harmful because it can be easily inhaled. So take precautions such as stay a metre away from the powdered enzyme. Also wear goggles to avoid contact with the eye as it could digest the carbohydrates that the eye is made up of. If swallowed: wash out mouth and give a glass or 2 of water. Seek medical attention as soon as possible. Solids can be mixed with 1kg of sand and placed in the refuse. Aqueous solutions (100ml) should be diluted in 10 litres of water and run to waste down the foul-water drain. As a general organic chemical [CG] unless kept in a refrigerator If solid or solution gets in eyes: flood the eye with gently running tap water for 10 minutes. Seek medical attention. If spilt on skin or clothes: remove contaminated clothing. Flood area with water and wash thoroughly with soap and cold water. If spilt in laboratory: scoop up as much solid as possible wash area thoroughly with detergent and water. Enzyme name Hazards Precautions to reduce risks Emergency procedures Storage and disposal Copper sulphate and Iodine This substance is harmful if swallowed and it has known to be an irritant if in contact with the eyes. It also irritates sensitive skin. It is also very toxic to the aquatic environment and may cause long-term adverse effects. The solutions that is equal to and greater than 1M should be labelled ?HARMFUL?. Wear goggles and you should handle the substance with care. Once finished using the substance pour down the drain and leave the tap on for about 2 minutes to reduce the environmental hazards. If swallowed: wash out mouth and give a glass or 2 of water. Seek medical attention as soon as possible. Solids can be mixed with 1kg of sand and placed in the refuse. Aqueous solutions (100ml) should be diluted in 10 litres of water and run to waste down the foul-water drain. As a general organic chemical [CG] unless kept in a refrigerator If solid or solution gets in eyes: flood the eye with gently running tap water for 10 minutes. Seek medical attention. If spilt on skin or clothes: remove contaminated clothing. Flood area with water and wash thoroughly with soap and cold water. If dusted inhaled then I would leave the lab to get fresh air. If breathing is slightly affected then should seek medical attention If spilt in laboratory: scoop up as much solid as possible wash area thoroughly with detergent and water. Results Inconclusive means that there was no result because the iodine didn?t stay brown after 12 minutes which is my limiting factor. Trial 1 concentration volume temp Time to reach achromatic point 0.01% Copper sulphate 2.5 cm3 40oC 500-520 seconds (510 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 2 concentration volume temp Time to reach achromatic point 0.01% Copper sulphate 2.5 cm3 40oC 480-500 seconds (490 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 3 concentration volume temp Time to reach achromatic point 0.01% Copper sulphate 2.5 cm3 40oC 500-520 seconds (510 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 1 concentration volume temp Time to reach achromatic point 0.02% Copper sulphate 2.5 cm3 40oC 80-100 seconds (90 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 2 concentration volume temp Time to reach achromatic point 0.02% Copper sulphate 2.5 cm3 40oC 580-600 seconds (590 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 3 concentration volume temp Time to reach achromatic point 0.02% Copper sulphate 2.5 cm3 40oC 560-580 seconds (570 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 1 concentration volume temp Time to reach achromatic point 0.03% Copper sulphate 2.5 cm3 40oC 620-640 seconds (630 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 2 concentration volume temp Time to reach achromatic point 0.03% Copper sulphate 2.5 cm3 40oC 640-660 seconds (650 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 Trial 3 concentration volume temp Time to reach achromatic point 0.03% Copper sulphate 2.5 cm3 40oC 620-640 seconds (630 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 1 concentration volume temp Time to reach achromatic point 0.04% Copper sulphate 2.5 cm3 40oC inconclusive 0.5% Amylase 5 cm3 1% Starch 5 cm3 2 concentration volume temp Time to reach achromatic point 0.04% Copper sulphate 2.5 cm3 40oC 700-720 seconds (710 secs) 0.5% Amylase 5 cm3 1% Starch 5 cm3 3 concentration volume temp Time to reach achromatic point 0.04% Copper sulphate 2.5 cm3 40oC inconclusive 0.5% Amylase 5 cm3 1% Starch 5 cm3 concentration % averages 0.01 503 seconds 0.02 416 seconds 0.03 636 seconds 0.04 1 Time taken to reach achromatic point X 103 710 seconds [image] [image]The formula I used to calculate the rate of reaction was: concentration % rate OF reaction 0.01% 1.988 0.02% 2.4 0.03% 1.5723 0.04% 1.408 Discussions For my 1st graph there was a general trend and correlation between the concentration of copper sulphate and the time taken for the solution to meet the achromatic point. This positive correlation was that the higher the concentration of copper sulphate that was added to the mixed solution, the higher the time taken for the amylase to break down the starch. The time taken for the solution to meet achromatic point was certainly higher by looking at these figures you could decipher that there is a high range between the 1st concentration tested and the last concentration tested: concentration % averages 0.01 503 seconds 0.04 710 seconds This evidence supports the idea that Copper sulphate inhibits the rate of reaction of Amylase. However I did get one anomalous result, on the concentration of 0.02% copper sulphate I got a higher rate of reaction than my 1st tested concentration. I think this is because I might of made up the dilution incorrectly, allowing the amylase to break down the starch at a faster rate. This brought down the average rate of reaction figure for that concentration, if I used just the two reasonable results I would have obtained the average figure of 580. This figure would have corresponded with all the other figures and with my hypothesis. For my Rate of Reaction graph there was a general trend and correlation of my results. The negative correlation means that the higher the concentration of copper sulphate the lower the rate of reaction will be. There is a basic trend of a negative correlation because copper sulphate is an inhibitor. More precisely Copper sulphate is a non ? active site directed inhibitor. Non-active site directed inhibitors bind strongly with enzymes tertiary structure breaking peptide bonds, as all enzymes are proteins. This alters the shape of the enzyme and its active site disabling the enzyme to function properly as the substrate is no longer able to fit into the enzymes active site. This means that copper sulphate probably bonded strongly with the amylase altering its tertiary structure which also alters its active site. As the active site has most likely changed (according to the lock and key theory) the starch may no longer fit in to the amylases active site as they are no longer geometrically compliant. Therefore there will be a lower concentration of working amylase enzymes so less successful collisions will take place. Therefore slowing down the rate of reaction. These two graphs suggest that my hypothesis of ?copper sulphate inhibiting amylase activity? is correct. I think that there were some limitations in my experiment that made my experiment less accurate and helpful to use. For example I was limited in the time I had to conduct my experiment, if I had more time I would have taken more readings for different concentrations to gain further results that may suggest my hypothesis was right. Another thing I would do if I had more time is that I would test other types of amylase such as fungal amylase and observe if copper sulphate had the same effect. I would also try thing such as test beyond the maximum concentration of copper sulphate to see if there is a peak in its inhibiting effect. Also I could have done in hind sight is to increase the range of the concentrations I used, for example instead of using 0.001, 0.002, 0.003 and 0.004, I could have used 0.001 0.003, 0.006 and 0.009 to receive a greater range of results. Another thing I would do if I had more time is that I would test other types of amylase such as fungal amylase and observe if copper sulphate had the same effect. The in accuracies that I?m worried that affected my results was that it was hard to detect the colour change in the iodine so my results I could?ve been carrying on with my experiment not knowing it had gone past the achromatic point. Now thinking about it I should have used a colourimeter to detect the colour change to make my results accurate. A colourimeter is more accurate as it shines a light through the solution detecting colour change. Luckily I didn?t get a lot of wide error bars showing that my results are still quite accurate. Error bars show the variability of all your repetitions and as my error bars were quite short u can see that most of my repetitions are quite close together. However the fact that there are error bars shows that there are flaws in my experiment and that is mostly due to the fact of trying to pin point the achromatic point. There is 1 very wide error bar in the 0.02% concentration, because the error bar is so big, this result cannot be used as it is inaccurate.

Investigating the Effect of Copper Sulphate on Amylase Activity 7.9 of 10 on the basis of 4097 Review.