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Enzyme Experiment

Enzyme Concentration and rate of reaction


Hypothesis:
1. Rate of reaction increases as enzyme concentration increase. As enzyme concentration increases, the number of molecules of the enzyme increases while the amount of substrate remains constant , the enzyme molecule collide more with the substrate, more collision means particles are more likely to reach activation energy, forming an enzyme substrate complex which catalyze the breakdown of substrate into products while the enzyme remains unchanged, Resulting in an increased rate of reaction. 2. Rate of reaction decreases as enzyme concentration decreases. As enzyme concentration decreases, the number of molecules of the enzyme decreases while the amount of substrate remains constant, the rate of collision between enzyme molecules and substrate decreases, less collision means particles are less likely to reach activation energy therefore less enzyme substrate complex is formed. The breakdown of substrate decreases and the reaction time also decreases, resulting in a slow rate of reaction.

Apparatus:
Milk powder suspension Test tubes Flat bottomed tubes Water bath Conical flask : for diluting enzyme Test tube holder Stop clock Standard acidified protease solution or alkaline trypsin solution Buffer 50cm3 pipettes, syringe or measuring cylinder 50cm3 beaker Eye protection PH indicator Gloves Glass rod Weighing scale

6 October 2012

Enzyme Experiment

Procedure:
Properly wash and rinse test tubes , measuring cylinder, conical flask, beakers , pipettes, glass rod and syringe with soap, tap water and distilled water Weigh 5g of powder milk on a weighing scale Measure 100cm3 of distilled water in a measuring cylinder Make milk suspension, by dissolving 5g of milk powder in 100cm3 of water Weight 0.5g of trypsin on a weighing scale Make 5ml of trypsin solution, mix 0.5g of trypsin powder in 100cm3 of water and add enough alkali to produce a pH of 9. Measure the pH of the trypsin solution using any suitable indicator With a glass rod, stir the solution properly to obtain an homogenous solution of milk Pipette 5cm3 of the milk suspension into a test tube(A) Pipette 5cm3 of the protease or trypsin solution into the test tube in A. Mix the solution thoroughly and immediately start the stop clock. Record the time taken for the protein solution to clear Make 0.2%, 0.4%, 0.6%, 0.8%and 1.0% concentration of enzyme by diluting the trypsin solution in 0.8%, 0.6%, 0.4%, 0.2% and 0.0% cm3 of distilled water. Repeat the experiment using different concentration of Enzyme given above. The reaction with 0.0% enzyme concentration serves as the control experiment.

Variables:
Independent o Volume of Enzyme concentration o Volume of distilled water Dependent o Time taken for the solution to clear Controls o Temperature: An increase in temperature would increase the rate of reaction, therefore making the result inaccurate also high temperature denatures the enzyme H o P of Enzyme: An Increase in pH would increase the rate of reaction, therefore making the result inaccurate also high pH denature the enzyme Volume of protein suspension Increase or decrease in protein suspension will increase and decrease the rate of reaction respectively, thereby reducing the accuracy of our Result.

6 October 2012

Enzyme Experiment

Errors:
Human o Reading measurement o Method of measurement Parallax error Reading accuracy of stop clock and indicator Using a weak alkali Identifying when the solution have completely cleared

To improve the validity, precision and accuracy of the result:


Repeat the experiment up to 5 times Avoid using anomalous result in my final analysis Use pH buffers to regulate pH of the enzyme solution Suspend the beaker in a water bath of 22*C to make the reaction a bit faster and provide the energy input to start the reaction. Use a digital stop clock to measure the time instead of analogue stop clock Use a digital pH indicator to measure the pH of the enzyme solution instead of litmus paper or universal indicator. Use the same quality of milk Plot a graph of my result Use micropipette instead of transfer pipette. The experiment should be performed on the same day. Different protein substrate should be used. Different Enzymes such as protease, pepsin .etc. should be used

Risk assessment and safety precaution


Alkaline or enzyme splashing on the skin or eyes o Use gloves o Use lab coat o Use eye protection o Avoid rubbing your eyes in case you have enzyme solution on your hands

6 October 2012

Enzyme Experiment

Overheating the solution o Use thermometer to measure the temperature of the water bath. Breaking apparatus o Handle apparatus with care o And provide extra pairs of each apparatus Increasing concentration of solution o Use a pH buffer

Results:
Enzyme conc. 20% 40% 60% 80% 100% Time 1 Time 2 Time 3 Average time 256.20 149.60 74.40 77.40 65.70 Rate of Reaction 1/time 0.00390 0.00668 0.01344 0.01292 0.01522 Temperature(*C)

265.8 139.8 93.6 80.4 67.2

247.8 150.6 68.4 0.59 0.51

255 158.4 80.4 74.4 64.2

22 22 22 22 22

Graph:

Enzyme Concentration and Rate of Reaction


0.01800 0.01600 0.01400 0.01200 0.01000 0.00800 0.00600 0.00400 0.00200 0.00000 0% 20% 40% 60% 80% 100%

Rate of reaction

Enzyme Concentration

6 October 2012

Enzyme Experiment

Trend in the Result:


As enzyme concentration increases, rate of reaction increases. The graph is steeper between 40%- 60% concentration The slope decrease between 62%- 80% concentration Between 20% - 39% and 82% - 100% concentration the slope are the same.

Explanation of the trend:


As enzyme concentration increases, rate of reaction increases. o As enzyme concentration increases, the number of molecules of the enzyme increases while the amount of substrate remains constant , the enzyme molecule collide more with the substrate forming an enzyme substrate complex which catalyze the breakdown of substrate into products while the enzyme remains unchanged, Resulting in an increased rate of reaction The graph is steeper between 40%- 60% concentration o The number of enzyme molecule is greater than the amount of substrate therefore more collision between enzyme and substrate, particles are more likely to reach activation energy as the collide frequently with each other, forming enzyme substrate complex which breaks down the limited amount of substrate at a faster rate, thus increasing the rate of reaction. The slope decrease between 62%- 80% concentrations. o This is due to human error in measuring the time of reaction. Because if concentration increases and rate of reaction decreases, it therefore means that the Enzyme have been denatured. In this case the rate of reaction between 82% - 100% concentration should be low but it was high! Implying there was an anomalous result and lots of errors. Between 20% - 39% and 82% - 100% concentration the slope are the same. o The rate of reaction remains the same because most of the substrate has been broken down between 82% - 100% and there are few molecules of substrate for the enzyme to break down. Between 20% - 39% the amount of substrate is more than the molecules of enzyme, as the concentration of enzyme is relatively low, therefore the reaction is slow.

The Highlighted values on the result shows the anomalous result which I did not include while calculating my average in other to make my results more precise and accurate.

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Enzyme Experiment

Conclusion
The purpose of this experiment was to determine the relationship or effect of Enzyme concentration and rate of reaction. The rate of reaction at different enzyme concentration increased this support the hypothesis that rate of reaction increases as concentration of enzyme increases. These suggest that the relationship between Enzyme concentration and rate of reaction is directly proportional. The graph of rate of enzyme concentration and rate of reaction also support this hypothesis and relationship.

6 October 2012