The Effect of Differing pHs on the Action of the Enzyme Pancreatin

The Effect of Differing pHs on the Action of the Enzyme Pancreatin
I predict that the optimum pH for the enzyme pancreatin is pH 9. I came to this decision after taking into consideration the following information. Catalysts speed up a reaction without being used up and so therefore they can be used over and over again. Due to them not being used up they are only required in very small amounts. Enzymes are biological catalysts and I will be using digestive enzymes for the experiment that I will be carrying out. The digestive juices contain enzymes, which break down large insoluble molecules into small soluble molecules. There are 3 main types of digestive enzymes and a different one is needed for each category of food. The enzymes which catalyse the breakdown of carbohydrates is carbohydrase; the enzymes which catalyse the breakdown of fats is lipase and the enzymes which catalyse the breakdown of protein is protease. Carbohydrates are broken down into glucose, fats are broken down into fatty acids (glycerol) and proteins are broken down into amino acids. Pancreatin contains a protease enzyme. Enzymes are all proteins and they are affected by temperature and pH; they have an optimum. Enzymes work on substances called a substrate.Enzymes have very specific shapes; each enzyme only works on its own specific substrates. This can be explained by using the lock and key theory where the lock is the enzyme and the key is the substrate. Only the correct shape and sized substrate (key) can fit into the active site (keyhole) of the enzyme (lock). The substance made at the end is called the product. [image] Diagram from www.elmhurst.edu/~chm/vchembook/571lockkey.html The enzyme is held into the very specific 3D shape by different types of bonds. If these bonds become broken or damaged in any way then the active site could change shape and so therefore the substrate would no longer bind as tightly. The conditions that damage the bonds are temperature and pH. Enzymes have an optimum for both of these factors. If the optimum pH for an enzyme was pH8 and the pH of the surroundings was altered slightly this would cause some of the ionic bonds holding the enzyme in its shape to break. The same also goes for temperature. However, the shape would only become slightly changed and so therefore the enzyme would still work but just not as quickly as it would in the optimum pH and temperature. On the other hand if the pH was altered a long way from the optimum this would then cause the shape to become more distorted and decreasing the chances of the substrate binding to the active site. This again also applies to temperature. When the substrate no longer fits into the active site we say that the enzyme has become denatured and so therefore can no longer be used in a reaction. This happens at high temperatures and extreme pHs. [image] The aim of my experiment is to find out the optimum pH for pancreatin. Conditions in the stomach are acidic (approximately pH2/3) because our stomach contains hydrochloric acid. There are two reasons as to why the conditions are like this 1. To kill any bacteria 2. It provides the correct conditions for the stomach protease. Food is the passed down into the duodenum (the 1st part of the small intestine) which is where the digestion of food occurs. Pancreatin is secreted into the duodenum to carry on the breakdown of food. Bile (which is made in the liver and stored in the gall bladder) is also secreted to neutralise the stomach acid and it also emulsifies fats. Bile is an alkali due to it containing sodium hydrogen carbonate NaHCO3 which is around pH10. Pancreatin itself is also slightly alkali. 41-16-Duodenum-L.gif Diagram from http://35.9.122.184/images/41- AnimalNutrition/html/source/32.html I carried out preliminary work to find the optimum temperature for pancreatin and my results showed me that the optimum temperature was 50?C. As a result of my preliminary work, I will be carrying out my experiment in water kept at the temperature of 50?C. I will control this by using a thermostatic water bath. For this experiment I will be using the following pHs 7, 8,9,10,11 because as stated at the beginning I predict that the optimum pH will be pH9 and so therefore I am doing a range of two pHs either side of this. In order to create the correct pH for the enzyme to work I will add a liquid called a pH buffer to the enzyme. A pH buffer is a liquid which is added to a liquid to make the solution the correct pH for my experiment. I will be using test tubes which will contain 7 cm³ of liquid in total. This will consist of 4 cm³ pancreatin and 3 cm³ of buffer. I will carry out the experiment three times for each pH so that I can then work out an average time. To show that the results are due to the enzyme being at different pHs I will use a control. My control will be that I will add a piece of film negative to boiled enzyme and if the negative doesn?t go clear then that proves that the enzyme is needed. [image] I will be using film negatives to find out what the optimum pH for pancreatin is. The film negatives are made up of gelatine (which is an insoluble protein), which is sandwiched between celluloid (transparent plastic) and the pigment. The gelatine acts to stick the pigment to the celluloid. During the experiment that I am going to carry out, the pancreatin will digest the gelatine and turn it into soluble amino acids. This will mean that the pigment will have nothing sticking it to the celluloid and so therefore the pigment will fall off. This will just leave the transparent piece of celluloid and so therefore I then know that the experiment has finished. Plan Apparatus 5 test tubes 1 Test tube rack 15 splints 15 Film negatives (35mm, 2 holes wide) Stop Clock Weights Scissors 6 Syringes 5cm³ Pancreatin (4 cm³ in each test tube) 5 pH buffers (3cm³ in each test tube) pH7 pH8 ph9 pH10 pH11 Thermostatic water bath-50?C Diagram Method 1. I will collect my apparatus. 2. I will use test tubes so that I can have 1 film negative in each test tube so that I can get more accurate results because I will only have to watch 5 film negatives at a time. 3. I will label the 5 test tubes with what pH they will contain (7, 8, 9, 10, and 11). 4. I will set up the 5 test tubes in a test tube rack. 5. In each test tube I will add 4cm³ of pancreatin. 6. In tube 1 I will add 3cm³ of pH buffer 7 using a 5cm³ syringe. 7. In tube 2 I will add 3cm³ of pH buffer 8 using a 5cm³ syringe. 8. In tube 3 I will add 3cm³ of pH buffer 9 using a 5cm³ syringe. 9. In tube 4 I will add 3cm³ of pH buffer 10 using a 5cm³ syringe. 10. In tube 5 I will add 3cm³ of pH buffer 11 using a 5cm³ syringe. 11. I will then put the test tube rack with the 5 test tubes in, into the thermostatic water bath. 12. I will allow 10 minutes before placing my film negatives into the test tubes. This is so the enzyme can acclimatise so that when I put the film negatives into the test tubes the reaction will always be taking place at the temperature 50?C. I will be using this temperature because from my preliminary work I found that 50?C was the optimum temperature. 13. I will then prepare the film negatives to be 35mm long and 2 holes wide. Each piece of negative will be cut from the same roll of film, so that each piece of film will have been exposed the same amount. 14. I will make a 1cm cut up the splints so that the negatives will slot in place and stay secure all throughout the experiment so that I will be able to check if the film has gone clear or not easily. 15. I will label each splint with the pH that it is going to be placed into. This means that I will be able to make sure that the film negatives do not get switched round. 16. After the enzyme has acclimatised for 10 minutes I will add the film negatives and start the stop clock. 17. I will not check the negatives for the first 2 minutes because from my preliminary work I found that nothing occurred. 18. After 2 minutes, I will check the film negatives every minute until they start to go clear. When there are signs of this happening, I will start to check the film negative every 30 seconds. 19. I will not check every 30 seconds from the very start or until some signs of a reaction taking place can be seen, because each time the film negative is taken out of the test tube no reaction can take place as the negative isn?t in contact with the pancreatin. 20. Once the film negative has gone totally transparent, I will record down the time that it reads on the stopclock. I will do this for each negative until all of the film negatives have gone totally transparent. 21. When this has happened I will replace the film negatives and repeat the experiment again for another 2 times so that I will have 3 sets of results for each pH. Therefore, I will be able to work out an average time. 22. When the optimum pH has been found, I will do the same experiment but including half values into the experiment. 23. The same method will apply for the half values, making sure that for each pH there are 3 sets of results so that again I can find an average time for each pH. In this experiment I am going to record in the results how long it takes for the film negative to go totally transparent. Fair Test Table Variable Why does it need to be controlled? How I will control it The temperature of the water bath Every enzyme has an optimum temperature, so therefore if the temperature changes, this could affect the results by either slowing or speeding up the reaction. I will be using a thermostatic water bath at the temperature 50? The Volume of Pancreatin and Buffer If there is more pancreatin, then there is more active sites so therefore the reaction will happen a lot faster. The buffer also needs to be equal or less than the amount of pancreatin. I will use a 5cm³ syringe so I get the exact amount of pancreatin and buffer that is necessary for the experiment that I will be carrying out. The Size of the Film Negative If the film negatives are different sizes then this will affect the results because there will be either be a greater or smaller quantity of gelatine. This will mean that either the reaction will occur quicker or slower affecting the time that it takes for the film negative to go totally transparent. I will be using the same size of film which will be 35mm long and 2 holes wide. As well as all the pieces of film negatives coming from the same roll of film so that they all have been exposed the same amount. Concentration of pancreatin If the concentration of pancreatin is greater then there will be more active sites and so therefore this will speed up the reaction. As well as if the concentration is smaller then there will be less active site and so therefore the reaction will be slower. I will use a 2% pancreatin solution throughout my experiment. The size of the splint If the split in the splint is larger then this will prevent the pancreatin react with the gelatine because the splint will be in the way. I will measure the splits in the splint with a ruler so that are as accurate as possible. Method I did exactly what I planned to do. Results Time taken for the negative to go clear/secs pH Trial 1 Trial 2 Trial 3 Average 7 499 536 579 538 8 478 525 274 525 9 454 494 542 496 10 457 505 554 505 11 20+ 20+ 20+ 20+ Time taken for the negative to go clear/secs pH Trial 1 Trial 2 Trial 3 Average 7.5 917 981 1024 974 8 543 628 546 572 8.5 870 907 966 1371 9 502 607 508 539 9.5 561 694 552 602 Conclusion From my results I can conclude that the optimum pH was pH9. I have concluded this because in both experiments pH9 took the lowest time for the negative to go clear. In my first experiment pH8 took 525 seconds, pH 9 took 496 seconds and pH10 took 505. From this data it can be said that pH10 on average didn?t take much longer than pH9 but this can also be said for pH8 however it did take slightly longer. In my second experiment where I investigated half values, I can again from my results conclude that pH9 took the lowest time for the negative to go clear. However from my results of my second experiment it can be seen that pH8.5 took the longest on average but this could be an anomalous result. It was expected that pH9 would be the optimum because of the conditions in the duodenum. Bile and pancreatin juices are secreted into the duodenum. Bile is an alkali due to it containing sodium hydrogen carbonate NaHCO3which is around pH10. Pancreatin itself is also slightly alkali. However acid from the stomach is also passed down into the duodenum causing the pH to be lowered slightly. I can also conclude from my results that pH took the longest. This could be because pH11 denatured the enzyme. When an enzyme is denatured bonds (what holds the enzymes specific shape) become broken or damaged causing the active site to change shape and so therefore the substrate would no longer bind as tightly slowing down the reaction. I agree with my prediction because I predicted that pH9 would be the optimum pH and my results confirm this. Evaluation Overall I feel that both experiments went well and I didn?t occur many problems. After analysing my results I feel that I can rely on them except for pH8.5 which I have regarded as an anomalous result because it took 1371 seconds for the negative to go clear which was even longer than pH8 which isn?t expected. During the experiments there were many elements that may not have been accurate. The splits in the splints were not all the same because even though I measured them some split even more when I cut them. If I was to carry out the experiment again I would use a Stanley knife instead of scissors because I could control the cut more easily. The size of the negative may not have been accurate because I cut them by freehand and so therefore I can?t be 100% sure that they were exactly the same size. To improve this I would not only measure it using the holes already on the negative but I would also use a ruler. Another factor is also that when I kept taking the negative out to see if it had gone clear I would have been preventing a reaction from taking place. As a results the overall time would be longer however I can?t do anything about this really because I have to check otherwise I wouldn?t know when the negative had gone clear. Another problem that I occurred is judging exactly when the experiment had ending because I couldn?t be a 100% sure that the negative had gone completely transparent and so I have taken this into consideration when looking over my results.

The Effect of Differing pHs on the Action of the Enzyme Pancreatin 9.9 of 10 on the basis of 4191 Review.