Introduction:
Enzymes are proteins that speed up many chemical reactions in cells. They act on substrates. Each enzyme acts only on specific substrates. Also it might be a reaction between a specific combination of substrates and a specific enzyme. Enzymes affect the rate of almost all chemical reactions which take place in organisms. Like all other proteins, enzyme function may be affected by a change in any aspect of its structure. The effect of a change in enzyme structure is called denaturation. Some substrates fit in the active site of the enzymes, but they are not taken apart by enzymes.They don’t let enzyme, which they occupied, take part in reactions anymore. They are called inhibitors.
The activity of enzymes is affected by pH, by temperature, presence of inhibitors, enzyme concentration and substrate concentration. There are still large areas of medical research in which the diagnostic potential of enzyme reactions has not been explored at all.
Catalase is an enzyme which is produced by every cell to break down hydrogen peroxide. Catalase is located in a cell organelle called the peroxisome. Hydrogen peroxide is a waste product of cellular activity that is poisonous to cells. However, it was used for a long time as a wound treatment (Some Eastern European countries still use it). Catalase speeds up the decomposition of hydrogen peroxide into harmless water and oxygen gas.
Dependent variable: Catalase enzyme activity (measured as % of oxygen produced)
Independent variable 1 experiment: pH 2 experiment: temperature
Controlled variable Amount of time
The purpose of this lab was to determine the effect of pH and temperature on the rate of catalase enzyme activity. Since the catalase speeds up the decomposition of hydrogen, the rate of catalase activity was measured indirectly by measuring the change in % oxygen produced over a fixed time period.
There were two experiments perfomed. First one determined the dependence of rate of catalase activity on the level of pH. In the first experiment our hypothesis was that if the level of pH decreases, then the rate of hydrogen peroxide degradation decreases. The second experiment investigated the effect of temperature on catalase activity. In the second experiment our hypothesis was that if the temperature decreases, then the speed of hydrogen peroxide decomposition decreases.
Materials and Methods:
Since hydrogen peroxide is potentially damaging to skin and eyes, goggles were worn for the duration of the experiment.
A Vernier® Oxygen Gas Sensor probe was connected to a Vernier Logger Pro® data collector, the apparatus was checked to confirm that it was working correctly.
200 ml of catalase solution was prepared by mixing 25 g of COOP™ yeast into lukewarm distilled water.
Experiment 1: Effect of pH on the rate of decomposition of hydrogen peroxide:
The first experiment investigated the effect of pH change on the rate of decomposition of hydrogen peroxide.
(Using a 2 ml measuring pipette), 5 ml of 3% hydrogen peroxide (H2O2) were measured into 3 25 ml test tubes (labelled pH4, pH7 and pH10).
5 ml of specific pH buffer (pH 4, pH 7 and pH 10) were added to each test tube.
The pH of the buffer/ H2O2 mixture was measured using universal pH indicator.
2 ml of catalase was added to the test tube labelled pH 4, the mixture was transferred into a closed chamber and a stopwatch was started. 30 s after transfer, measurement of % oxygen was started using the Vernier® apparatus. Data collection was continued for 180 s. This procedure was repeated for the test tubes containing hydrogen peroxide/pH 7 and pH 10 buffer. Data was saved and transferred to EXCEL for the purpose of data analysis.
Experiment 2: Effect of temperature on the rate of decomposition of hydrogen peroxide:
The second experiment investigated the effect of temperature change on the rate of decomposition of hydrogen peroxide.
An ice bath was prepared and maintained at 0 oC. 3% hydrogen peroxide, catalase solution and distilled water were maintained in the ice bath. Temperature was confirmed using a thermometer.
Similarly, a hot water bath was prepared and maintained at 60 oC. 3% hydrogen peroxide, catalase solution and distilled water were maintained in the hot water bath. Temperature was confirmed using a thermometer.
(Using a 2 ml measuring pipette), 5 ml of 3% hydrogen peroxide (H2O2) were measured into 3 X 25 ml test tubes (labelled 0 oC, 22 oC and 60 oC).
5 ml of distilled water were added to each test tube.
2 ml of catalase was added to the test tube labelled 0 oC, the mixture was transferred into a closed chamber and a stopwatch was started. 30 s after transfer, measurement of % oxygen was started using the Vernier® apparatus. Data collection was continued for 180 s. This procedure was repeated for the test tubes containing hydrogen peroxide/22 oC and 60 oC. Data was saved and transferred to EXCEL for the purpose of data analysis.
Discussion:
In Experiment 1 it was observed that any change in the level of pH (both decrease and increase) affected the speed of breakdown of hydrogen peroxide. In Experiment 1 was observed that both decrease and increase of the level of pH increased the speed of hydrogen peroxide breakdown. However, the rate of oxygen production was higher at the pH level 10 than at the pH level 4. Without any catalase or hydrogen peroxide,no oxygen was produced, since there was no t hydrogen peroxide breakdown. Our hypothesis was refuted. We assumed that the higher the pH, the faster the rate of hydrogen peroxide breakdown. However, the patterns which were assumed were not obeserved in the experiment. After the experiment was made a conclusion that any change in pH speeds up the produce of oxygen by breakdwon of hydrogen peroxide, but the increase in pH speeds up the breakdown process higher, rather than decrease.
In Experiment 2 it was observed that any change in temperature negatively affects the speed of hydrogen peroside breakdown Not exactly – a change in temperature affects the rate of hydrogen peroxide breakdown. The rate of decomposition of hydrogen peroxide at room temperature was much greater than at 60 Co and 0 Co. It was assumed before the experiment that the lower the temperature, the lower speed of hydrogen peroxide decomposition. Our hypothesis was partly upheld by experimental data.. However, it was observed that any great change in temperature negatively affects enzyme catalase activity, since at these temperatures enzyme denatures.
Error sources:
In both experiments, more precise and accurate apparatus should be used to measure the volume of liquids (pH solutions, yeast, hydrogen peroxide), to control the ratio of liquids and the purity of the. The measuring burette would perfectly fit.
The oxygen measuring apparatus also should be improved,. The devices were not calibrated before the experiment began, andtheir accuracy was not confirmed. Also it was unknown whether these devices were calibrated. These factors could affect the data that was collected.
In Experiment 2 to control the temperature of catalase and hydrogen peroxide apart from water and ice bath, the temperature of the environment (room where exepriment was performed) should also be controlled, as the catalase and hydrogen peroxide, in order to improve the relaibility of the results obtained.
Also in each, there was a 30 second delay before oxygen measurement began. . As human error factor s are present in every experiment, there might be a mistake of 1-2 seconds, which may have affected the results of the experiment.
Conclusion:
In experiment, 1 was observed that a change in pH alters the rate of decomposition of hydrogen peroxide. The optimal pH for cyeast catalase activity appears to be pH 10. It ust be recognized, howebver, that at extreme pH levels (pH 1-2 and pH 13-14 ), the enzyme denatures and the speed of hydrogen peroxide breakdown decreases. In experiment 2, extremes of temperature reduced the rate of hydrogen peroxide breakdown. It happened, because at these temperatures hydrogen peroxide denatures. In fact, at low temperatures the enzyme should not be denatured, but the reaction rate would be slow since the rate of enzyme-substrate collisions would be slow.
This is a different font to the rest of the document.
References:
• 27 July 2004, SEPS. Catalase FAQ.
• Repose, D. 2008. Introductory Biology at UNCG. Writing a lab report.
• 2009, MHS Science. Sample Lab Report: Factors which Affect the Acivity of the Enzyme Catalase
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