Maryam Jamshidi (Core practical)Activity - Enzyme concentration and Enzyme activity Source: http://mycourses.mycandi.ac.uk/pluginfile.php/237165/mod_resource/content/0/01_core_practicals/Activity_2.11_Enzyme_concentration/Notes%20for%20writing%20up%20Enzyme%20core%20practical%20_v1.pdf

Purpose: to investigate the effect of the Trypsin concentration on the rate of reaction on breaking down Casein, the protein found commonly in milk. Introduction Enzymes are proteins that speed up particular reactions within cells. They are biological catalysts as they take part in a reaction yet remain unchanged at the end of the reaction, and although they have an impact on the rate, they do not affect other components within the reaction. Most catalysts are not selective as they catalyse more than one reaction. Enzymes however, are highly selective and catalyse one specific reaction only. This is due to the tertiary structure of the protein which generates the specific shape of an enzyme molecule. A lot of enzymes consist of a non-protein (cofactor) and a protein (globular) component. Inappropriate temperatures and pH are able to denature proteins, which means the polypeptide chains can be impacted, which can lead to a change in shape, causing denaturing to take place for the enzyme. The optimum conditions for an enzyme to work are those conditions that allow it to work at its fastest rate. Different enzymes have different optimum temperatures and pH levels, therefore, the catalytic activity within enzymes are pH and temperature sensitive.How enzymes work For two molecules to react they must collide with one another in the right direction (orientation) and with sufficient energy. Sufficient energy means that between them they have enough energy to overcome the energy barrier to reaction. Lock and key theory:In this theory, the substrate fits into the active site of the enzyme to form an enzyme-substrate complex. Bonds break while the substrate is locked in the enzyme-substrate complex, which will allow the product to be produced. Induced fit theory: In this theory, the enzyme molecule changes shape as the substrate molecules gets close. The change in shape is 'induced' by the approaching substrate molecule. This model relies on the fact that molecules are flexible because single covalent bonds are free to rotate. The product of these theories is a broken down version of the substrate, in other words, when bonds break within a molecule, the molecule breaks apart to even smaller molecules, where it will be able to be absorbed into cells.Hypothesis: The higher the concentration of enzyme, the faster the rate of the reaction of the trypsin breaking down the casein. Variables: The independent variable of this experiment is the concentration of the trypsin enzyme as water was added to dilute the enzyme concentration in controlled amounts. The dependent variable of this experiment is the time taken for the solution to reach 50%T in the colorimeter. The control variables of the experiment are the types of enzymes used (the trypsin and casein), the volume of the solution in the test tubes as it was always at 5cm, and the volume of milk put into the solution was always at 3cm. Method: Apparatus:1. Beaker of casein solution1. Beaker of trypsin solution1. Beaker of distilled water at pH91. 6 x small glass Test tubes,1. Test tube rack1. Stop clock1. 2 x 5 cm3 syringe1. 1 cm3 syringe1. Colorimeter

I started off by preparing my apparatus. I filled my first test tube with 5cm of the trypsin solution, the second with 4cm of the trypsin solution and 1cm of water, the third with 3cm of trypsin solution and 2cm water, and so on until no volume of trypsin was used in any more test tubes. I then made a solution of 5cm of trypsin, 3cm of water, which is what I used as a reference for my colorimeter. I then labelled each test tube so I wouldnt mix them up during the experiment and so it would be easy to identify the different concentrations throughout the experiment. I turned on the colorimeter and put in the reference test tube as I pressed the R button. I then took it out, and put one of the test tubes into the colorimeter. I pressed the graph button, then put 3cm of the milk solution into the test tube, and pressed %T until it reached the reading of 50%T. I began the stop clock as soon as I put the milk into the test tubes to be able to see the rate of reaction in seconds. I did this for all concentrations, including the solution without the trypsin enzyme in it.

Results and Conclusion:time to 50%T / srate of reaction (1/t) /s-1

Enzyme conc. / gdm-3123456789meanSDmean SD

1182312/17.915131724.351840.0570.0143

21213.34.2/13.2761318.761150.0910.0403

3612.24.110.48.811.5/814.88840.1210.0641

44.59.13.55.37.7636710.17620.1600.0628

53.58.783.26.56.062.57155.9520.2120.1065

Through my class results I can see that as less water is used to dilute the trypsin solution (so the stronger the concentration of the trypsin) the less time it takes for the trypsin to break down the casein molecules in the milk mixture added to the enzyme solution.

Interpretation:It seems that the higher the trypsin concentration, will mean that there is more trypsin enzymes within the given volume, which will allow the casein to hit an active site of an enzyme more likely than a diluted solution as there are more trypsin molecules available compared to water molecules.

Evaluation:According to the standard deviation, there appears to be a very wide range in the time taken for the trypsin to react for each individual group. This makes the data unreliable as the standard deviation shows that there are likely to be anomalies in the results. Accuracy was not able to be measured properly as the experiment wasnt repeated, instead, as a class it was done once by each group, and results were put together to show a range of the results. People tried to be very accurate, to make a fair class result, but although precision was said to be carried out, it cannot be proved and therefore not is not very reliable.The syringe doesnt very accurately measure the volumes of water and trypsin required and also can gather bubbles within, which will affect the overall volume of the solution. The stopwatch may have not been too accurate as time is wasted to start and stop the clock, which has an impact on the rate of reaction that we write down. 1