Investigating Catalase Activity in Different Plant Tissues.
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Investigating Catalase Activity in Different Plant Tissues By Dariush Mahboubian Aim In this experiment I am going to investigate the effect of pH on the activity of the enzyme catalase. I will be using plant extracts to obtain the catalase for my experiment. The plant material available is carrot, radish, potato and spring onion. In my preliminary experiment I will be looking at which of these materials will be best suited to my main experiment. In my main experiment I will be measuring the rate of reaction by finding out the time taken for a certain amount of product to be given off. The rate of reaction is basically the same as 1/time. I will do each experiment and repeat it 3 times to get an accurate average result. Background Science During respiration plants and animals make hydrogen peroxide. Hydrogen peroxide is poisonous and they need to get rid of it as soon as they produce it. They get rid of the hydrogen peroxide using the enzyme catalase. Enzymes are biological catalysts which speed up chemical reactions without being changed at the end of the reaction and without being used up. They are made from protein so they are quite delicate, because the tertiary structure of proteins is held together by very weak bonds like hydrogen bonds and hydrophobic bonds. Catalase catalyses this chemical reaction. Many scientists have studied the way enzymes work. The scientist Fisher developed the lock and key hypothesis by saying that enzymes have an active site with a precise shape, and that only substrates with the same shape as the active site could fit inside. Once inside the enzyme the substrate can be broken down to form products. Then Koshland took the theory a bit further. He said that the enzyme's active site wraps around the substrate, so the lock and key hypothesis is only really true once the substrate is actually inside the enzyme.
I will store the solution away from heat and light and avoid contaminating the solutions with substances that could speed up the breakdown of hydrogen peroxide, leading to pressure build up. Acid and alkaline buffers Corrosive: burns the eyes and skin I will only be using small volumes and I will wear safety glasses and a laboratory coat. Catalase enzyme contained in yeast Low hazard: can be a sensitiser to allergens I will only be using a low concentration inside yeast cells and I will wear safety glasses and a laboratory coat. Equipment list Equipment used Reason for use Fresh carrot Highest catalase activity of all the plant specimens tested. Serrated knife To cut off and skin a small piece of carrot from a whole carrot. Razor blade To cut each piece of carrot precisely to a volume of 1cm3. Ruler To measure each piece of carrot precisely to a volume of 1cm3. White tile To stop the blades damaging the laboratory table. Distilled water To rinse the pipette. pH7 buffer To keep the reaction mixture at a constant pH7. Pestle and mortar To extract the catalase by pounding and grinding. PONY vial Airtight container to store fresh catalase. 2 10ml syringes Precise way of measuring out a volume of buffer and H2O2. Stopwatch Precise way of measuring time taken. Spatula To scrape the carrot from the mortar into the PONY vial. Filter paper discs To adsorb the carrot solution. Filter paper To blot the filter paper discs onto. Permanent marker To label the PONY vials. Boiling tubes For adding buffer to the carrot solution. Forceps To dip the filter paper discs into the carrot solution. Glass well For reaction to take place. Method 1. I took a white tile and cut the outer skin of the carrot using a knife. 2. I used a razor blade to cut the piece of carrot into a cube with sides of 1×1×1 cm3 with the help of a ruler.
A way to improve the reliability of the experiment would be to use the same type of mould for each practical, for example a potato borer. My 3rd main source of experimental error: In different practical sessions the carrot was ground up in different ways, if it was ground up a lot more, maybe more catalase would get let off. It was also difficult to scrape all the carrot paste into the PONY vial and different amounts might have been left in the two different practical sessions. My 4th main source of experimental error: To measure out volumes of buffer and hydrogen peroxide solution, I used two 10 ml syringes. These are quite accurate but there are more accurate apparatus like burettes. My 5th main source of experimental error: Some of the filter paper discs were quite flat but some of them were quite bent, this could affect how aerodynamic they are when it comes to rising to the surface of the hydrogen peroxide solution. One way to improve this would be to iron the discs so that they are all equally flat. My 6th main source of experimental error: When you put each disc in a PONY vial, the sediment gets disturbed, changing the amount of catalase so making the experiment unfair and reducing the reliability of the experiment. This could be improved by leaving the sediment to settle for a long time, then just pouring off the upper liquid part into another PONY vial. My 7th main source of experimental error: The stopwatches are quite accurate but people's reaction time is never the same and also changes during the day. Some people are a lot more alert in the morning than the afternoon, so if they do a practical in the morning their reaction rate will be faster than their reaction rate in the afternoon. This will change if accuracy of the results if the sessions are done at different times of the day. This probably wasn't very significant because we did both practical sessions in the morning.
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