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Investigating the effect of pH on the activity of an enzyme.

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Saira Hamid AS Biology ByB3(b) coursework. Investigating the effect of pH on the activity of an enzyme. Planning. In this investigation the effect of pH on the activity of protease on exposed photographic film is to be tested. In order to do this the colour change of the test solution will have to be measured, this can be done by measuring the absorbency by using the colorimeter. The results obtained will be from 0.00 being the lowest value; there is no fixed highest value, as this depends on the colour change of the test solution. The experiment will be carried out by reacting the enzyme with the substrate. The enzyme is the protease which is mixed with the pH buffer solution; the substrate is the exposed photographic film. No reaction occurs unless the substrate is present, this will be placed in a water bath and removed after a certain amount of time and colour change will be recorded and absorbency will be measured. Variables. There are many variables in this experiment but the 2 main variables that concern this experiment are: * The independent variable, which is the pH buffer solution, this is the variable that is going to be changed from various pH values between 1-14. * The dependant variable is the absorbency as this is going to be measured, the lowest value is 0.00, the blank solution is most likely to give this result, and is used to zero the colorimeter, but the higher the number the more effect pH has on the activity of protease. The other variables have to be kept controlled and constant so that they don't affect the result. These variables are: > Volume of pH buffer solution. > Volume of protease. > Amount of time for reaction to occur. > Temperature of water bath. > Same size exposed photographic film. * Volume of pH buffer solution: if this is not kept constant for all buffer solutions throughout the experiment, then the experiment will not be fair. ...read more.


* To each of the buffer solution measure out 2.5cm� of protease and place this in each test tube. * Place these test tubes in a water bath at 37�C until they are all at the same temperature. The water bath is already pre-heated to the right temperature in the incubator water bath. * Now place the photographic film in the test tubes using tweezers. The tweezers are used instead of using fingers as this could cause contamination and start reaction before time. At the same time as placing the film in solution start a stop watch and incubate for 20 min. * When test tubes have been incubated for 20 min remove the photographic film as this stops the reaction. * Whilst waiting for this reaction to occur carry out the control. * To prepare the control pour 2.5cm� of a buffer pH of your own choice into a test tube and place 2.5cm� protease and place in a water bath of 70-80�C * Remove this test tube from water bath once the test tube has reached the temperature of water bath. * The purpose of this control is to see if there is and activity without photographic film. * So when all test tubes are out of there water bath lave them to cool for 10 min before measuring the absorbency in the colorimeter. * Check that each cuvette is of same type and shake substance in test tube and pour equal amounts of the test solution in a cuvette. * Before placing the test solution in the colorimeter check that the colorimeter reading is set to absorbency. * Before testing a solution the colorimeter must be zeroed this done by using a small amount of buffer and protease mixed together which has not been place in a water bath. * Test solutions by placing the cuvette in the colorimeter and remove record reading and zero the colorimeter before next cuvette is inserted. ...read more.


* The experiments should have been repeated in the same day as conditions were more likely to be the same. * The amount of reaction should be increased to about the 30 min as it gives it more time for reaction to occur. * Making sure the number of colorimeter was recorded in order to obtain accurate results. Overall even though I encountered various problems I always found a way to improve them, and ensure that the experiment was carried out as accurately as possible. I followed all the steps of accuracy as above and my results were reliable as I had no anomalous results so therefore my experiment must have been precise and accurate up to a certain extent. Also according to my analysis the graph is also a smooth curve. Also my analysis said that the optimum pH is 2 this is correct as I have supported this with scientific knowledge, this must be true because another example of where pH effects the activity of an enzyme is in the stomach during digestion when proteins have to be broken down by pepsin but it is turned into its active form under high acidic conditions as it can only work under these conditions, for the reason that after pH 2 pepsin becomes denatured and inactive. The apparatus did have its limitations but most of these limitations could not be prevented. The majority of the mistakes were made whilst doing the experiment the first time but this cannot be helped as I have not done this experiment before, so the mistakes I made during the first time were corrected when carrying out the repeats. The techniques which were used in this experiment were of high quality especially in the repeats everything was carried out with care although sometimes thing had to be rushed slightly in order to get the experiment finished, but this was not too bad it just meant working at a faster speed but also taking care. In my experiment I did not encounter any breakages or spillages. So this did not hold me back or stop me from working. ...read more.

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