AP BIOLOGYLAB REPORTEXPERIMENT 1 – INTERACTIONS-ENZYME ACTIVITYGroup 5Bin Islam10/11/18Introduction:Enzymes are biological catalyst. Which speeds up chemical reaction in our body. They (enzymes) do that by forming enzyme-substrate complex.
For that to form their shape is very important. Every enzymes have different shapes thus form enzyme-substrate complex with different substrate. Enzymes shape determines whether it can form enzyme-substrate complex with the substrate or not. Enzyme works best in certain environmental condition. Which is their optimum environment.
However enzyme activity can be affected by many factors. They are temperature, pH, ionic concentration and substrate concentration. If temperature and pH are too high than their optimum temperature and pH, then the enzyme activity will decrease and it will denature. The optimum environment is determined by where the enzymes are found in the organism.If the ionic concentration is increased the ions with bind to ionic side chains of protein and make it easier for substrate to locate, therefore change the rate of reactions and if it decreases then its vise versa. If the substrate concentration increases then the rate of reaction will increase as there will be so many substrates for enzyme to bind with and if decreases then vise versa.
In the experiment we are looking at how different factors affect the rate of reaction of enzyme peroxidase and the substrate hydrogen peroxide. Peroxidase breaks down peroxide. For this we will perform 2 experiments. One will be about how the pH levels affect the rate of reaction of peroxidase and the other will be how the concentration of substrate hydrogen peroxide affect the rate of reaction of enzyme peroxidase.Hypothesis:I predict for the first experiment that the optimum pH of the enzyme activity is pH of 7. So the pH close to 7, in pH 5,6,8 will have the enzyme peroxidase will have faster rate of reaction than in pH 3,10,12. For the second experiment the hydrogen concentration of 0.
20, 0.50 and 1.00% will have faster rate of reaction with peroxidase than 0.01% concentration. When it becomes saturated the reaction will stop.
Procedure:Procedure 1- BaselineMaterialsTurnip Peroxidase0.1% hydrogen peroxideGuaiacolDistilled waterPeroxidase Color Chart3 test tubes (Approximately 16×150 mm) and appropriate test tube rackTimer1,5,and 10 mL graduated pipettes, pipettes pumps, or syringes (1,2,5, and 10 mL)Step 1 Using two of the three test tubes, mark one as “substrate and the other as “enzyme”. Then for substrate tube add 7 mL of distilled water, 0.3 mL of 0.1% hydrogen peroxide, and 0.2 mL of guaiacol. Which makes it a total of 7.
5 mL. After that cover the test tube with parafilm and gently mix.Step 2 Now for enzyme tube add 6 mL of distilled water and 1.5 mL of peroxidase for a total volume of 7.5 mL to the enzyme test tube.
Cover the test tube with parafilm and gently mix.Step 3 After that combine the contents of the two test tubes in the third test tube, cover the test tube with parafilm and invert twice to mix. Place it back in the rack and immediately start timing the reaction.Step 4 Observe the color change for next 5 minutes and rotate the tube before each reading. Record the color change at 0,1,2,3,4,and 5 using the color chart.
(You can take pictures to record the color changes)Step 5 Graph your data in your notebook.Procedure 2- Determining the effect of pH on enzyme activityMaterialsTurnip Peroxidase0.1% hydrogen peroxideGuaiacolBuffers with pH levels- 3,5,6,8,10,12Distilled waterPeroxidase Color Chart12 test tubes (Approximately 16×150 mm) and appropriate test tube rackTimer1,5,and 10 mL graduated pipettes, pipettes pumps, or syringes (1,2,5, and 10 mL)Step 1 Using clean test tubes , make 6 pairs of original substrate and enzyme tubes for total of 12 tubes or 6 pairs. Label the tubes with the pH 3,5,6,8,10, and 12. And substitute the distilled water with the pH buffer of the label.
-For each substrate tube add 7 mL of distilled water, 0.3 mL of 0.1% hydrogen peroxide, and 0.
2 mL of guaiacol. For a total of 7.5 mL-For each enzyme tube add 6 mL of specific pH solutions which are 3,5,6,8,10, and 12 (goes with the labeled pH tubes) and 1.5 mL of peroxidase for a total volume of 7.5 mL. For example in the enzyme tube of first pair you labeled it as 3.
So you substitute the water with buffer solution of pH 3. In enzyme tube of second pair you labeled it as 5. So you substitute the water with buffer solution of pH 5.Step 2 Combine the substrate and enzyme tubes of all six pairs in the substrate tube, cover it with parafilm, gently mix and put it back on the rack. Immediately begin timing the reaction.
Step 3 Record the color change at 0,1,2,3,4 using the color chart.Step 4 Graph your data as color intensity vs pH.Procedure 3-Determining the effect of substrate concentration on enzyme activity.MaterialsTurnip Peroxidase1% Hydrogen peroxideGuaiacolDistilled waterPeroxidase Color Chart6 test tubes (Approximately 16×150 mm) and appropriate test tube rackTimer1,5,and 10 mL graduated pipettes, pipettes pumps, or syringes (1,2,5, and 10 mL)Dilution You need to use dilution to get the amount of hydrogen peroxide concentration.
We will be using 0.20%, 0.50% and 1.00%0.20%- To get 0.20% you use the appropriate pipettes to take 2 mL of 1% hydrogen peroxide and mix it with 8 mL of distilled water.0.
50%- To get 0.20% you use the appropriate pipettes to take 5 mL of 1% hydrogen peroxide and mix it with 5 mL of distilled water.1.00%- For this one you don’t have to dilute. Since you already have 1% hydrogen peroxide.Step 1 Using clean test tubes , make 3 pairs of substrate and enzyme tubes for total of 6 tubes or 3 pairs.
Label each substrate tubes with the hydrogen peroxide concentration 0.20%,0.50%, and 1.00%. For substrate tube add 7 mL of distilled water, 0.3 mL of labeled hydrogen peroxide, and 0.2 mL of guaiacol.
Which makes it a total of 7.5 mL. For enzyme tube add 6 mL of distilled water and 1.5 mL of peroxidase for a total volume of 7.5 mL to the enzyme test tube.
Cover the test tube with parafilm and gently mix.Step 2 Combine the substrate and enzyme tubes of all six pairs in the substrate tube, cover it with parafilm, gently mix and place it back on the rack. Immediately begin timing the reaction.Step 3 Record the color change at 0,1,2,3,4 using the color chart.
Step 4 Graph your data in your notebook. -Turnip Peroxidase Color Chart- Data:Procedure 1- BaselineProcedure 2- Determining the effect of pH on enzyme activity-For the graph of procedure 2 you can not see the line of pH 3 and pH 10 because it is underneath the line of pH 12. Since, they all have the same values.Procedure 3-Determining the effect of substrate concentration on enzyme activity.Analysis:Procedure 1- BaselineThe baseline was our control group to compare the changes caused by our independent variables in procedure 2 and 3. The data for baseline shows how the color change/reaction will occur without any independent variables. We can see in the data that the rate of reaction is high when there were no independent variable affecting the enzymes rate of reaction.
We can deduce that after 1 minute the color changed to 5 and after 1 more minute it jumped to color 7 in 3 minutes the color changed from 1 to 8 and in the end of 4th minute the color changed to 9 and at 5 it remained the same which shows that this is the highest color level in the reaction. This is a control group and it showed high rate of reaction with no factors affecting it.Procedure 2- The effect of pH on enzyme activityProcedure 2 was used to show the effect of one of the environmental effects which was pH, on the enzyme activity. In the data we can see that the enzymes doesn’t react at all at pH level of 3, 10 and 12. And the pH of 5 and 6 had a similar rate of reaction as the baseline.
For pH 5, after 1 minute the color changed to 5 and after 1 more minute it jumped to color 8 (the 2nd minute) the color remained the same (8) for next 2 mins. Which tells us that the highest color change for pH 5 was 8. For pH 6 it was the same as ph 5 but the only difference was that the color changed to 9 after the last/4th minute. Which tells us that the highest color change for 6 is same as the highest color change for baseline and we can see that the rate of reaction was similar too. For pH 8, at the end of 1 minute the color change was 4 and after the 2nd minute it was 6 and remained the same until the next minute, after 3rd minute the color started to change and at the end of 4th minute it reached color change of 7.
Which tells us that the highest color change for pH 8 was 7. These data support my hypothesis as the rate of reaction after adding buffer pH near 7 which were 5,6 and 8 was close to the rate of reaction of the baseline.Procedure 3-The effect of substrate concentration on enzyme activityThis experiment was conducted to determine the effect of substrate concentration on enzyme activity.
We will use the control group or baseline to see the effects of substrate concentration. The data shows us the rate of reaction (color change) at different concentration. In the data we can see at the substrate concentration of 0.20% the rate of reaction was fairly lower than the rate of reaction of 0.50% and 1.
00%. In 0.20% the highest color change was 5 whereas the highest color change for 0.50% was 6 and for 1.00% was 8. For 0.20% and 0.
50% it was saturated, the reaction stopped after the 3rd minute. The data supports my hypothesis mentioned earlier as it clearly shows the increase in substrate concentration increased the rate of reaction (Color change).There is an error in the data. The error is that we didn’t time until the reaction was saturated for 1% hydrogen peroxide. This error decreased the amount of data we collected and therefore decreased the amount of evidence to support our hypothesis. Some error done during the experiment could have resulted the data of 1% hydrogen peroxide. The data looks like an unusual data as there were no saturation and it kept increasing (color change).
One source of error could be making a mistake in measuring. When we were using the pipettes for 1% hydrogen we were in rush cause of lack of time. That time we could have made mistake in measuring, which could have affected the data and resulted in the data we have.Conclusion:The experiment shows how the enzyme activity is affected by different factors. Which were pH and Substrate concentration. We can prevent future error by being careful about the time and manage it accordingly.
Also be careful while measuring. Another suggestion is to use a higher variety of concentration for hydrogen peroxide to give us more variability for our data and time it longer. For future experiment you could test different enzymes from different environment to compare how the environment of the enzymes affect its rate of reaction or how their optimum pH is determined by their environment (where the enzyme is present).