Each type on enzyme has its own range of pH in which it functions to its optimum. For example intercellular enzymes have an optimum pH of 7 that is neutral conditions. Strongly acidic or alkaline conditions denature enzymes.
Intracellular enzymes such as digestive enzymes work best in acidic or alkaline conditions. For example the protease enzyme pepsin works best in acidic conditions where it is found in the stomach that has a pH of 2. If the pH was increased the pepsin would denature and would not be able to hydrolyse fats. Trypsin found in the duodenum has an optimum pH of 8.5 and works effectively in alkaline conditions. If the pH was decreased the trypsin would denature and have no effect on the substrate molecule.
Method
The aim of the experiment was to identify the affects of different pH on enzymes. The time taken to collect oxygen was measured in seconds between the reactions of peroxide in different buffers of yeast.
2H2O2 + yeast -> 2H2O + O2
Before touching any of the solutions and equipment safety precautions was taken. Safety goggles, lab coats and gloves were used as hydrogen peroxide is corrosive and an irritant that can burn skin and eyes.
Test tubes were placed on a rack and labeled pH2, pH4, pH7 and pH9 as this was the pH of the buffer solutions. In each test tube 3ml of yeast was added to each one using a pipette. 1ml of different buffer solutions was added to the test tube according to the label. For each buffer solution a different pipette was used so as not to contaminate the yeast and affect the results. Once the test tubes were ready the rest of the apparatus was prepared in the standard format shown below.
A large jug was filled with cold water and a 30ml gas syringe was placed inside and filled with water to the top. The tap on the gas syringe was fastened with a clip to prevent water from escaping. One end of the oxygen collecting tube was placed securely beneath the gas syringe in the water. The other end of the gas collecting tube was connected to a bung. The bung also had a needle all the way through that connected to a syringe. The bung attached to the test tube and prevented gas escaping through the top of the test tube and allowed it to pass through the gas collecting tube. The syringe was filled with 1ml of peroxide and then carefully screwed onto to the needle. A stopwatch was used and started as soon as the peroxide was forced out of the syringe and into the different buffers of yeast. The oxygen formed from the reaction travels through the gas collecting tube and collected in the gas syringe. When 10ml of gas was collected the stopwatch was stopped and the time in seconds was recorded in a table prepared before hand.
The bung was then taken off cautiously due to the needle sticking out of it and placed onto the next test tube. The gas syringe was filled with water again and clipped shut firmly. The syringe above the bung was filled with 1ml of peroxide yet again and the time taken to collect 10ml of oxygen was recorded. These steps were repeated twice more on different test tubes that contained different buffers.
Results
Conclusion
The optimum pH for the enzyme is pH 9 as the rate of reaction increases. The yeast worked better at neutral conditions compared to acidic as it collected oxygen in a faster time. The enzyme yeast did not favour pH 2 as the rate of reaction was slow probably due to the fact that many of the enzymes in the reaction had denatured. For pH 9 rate of reaction was fastest as the enzymes had not denatured and working to their optimum.
Evaluation
As this was the first time using the apparatus and measuring the time to collect 10ml of oxygen, I found it slightly difficult. However after a few practice trials I gradually understood the method and carried out the experiments easily.
The results produced were not reliable as the results produced are of only one set of experiment. To produce accurate results the experiments should have been completed once more so as to get an average from them. Also doing the experiment again would have shown if the first set of results were reliable.
The syringe attached to the needle was problematic as it was difficult to detach it from the needle delicately and it was also hard to compress the peroxide into the test tube of yeast to start the reaction. When the peroxide was being pushed out of the syringe it was important to screw the syringe in compactly as otherwise not all the peroxide would be used in the reaction making the results inaccurate.
In this experiment the tap on the gas syringe had to be firmly closed by the hands to prevent water from escaping, which was difficult as the pressure used on the tap was unintentionally loosened as times or the tap was held too firmly.
To overcome these problems the next time the experiment is done more experience would be acquired so it would be done more efficiently and repeated twice more to gain reliable results. When repeating the experiment a clip will be used for the tap on the gas syringe. Instead of preparing all the test tubes at once they would be prepared individually before starting the reaction, so that the buffer would spend a similar amount of time in the yeast to other buffer solutions.
Experiment was done starting with pH2 to pH9 as it was predicted that the enzyme activity would be low during the start and gradually increase to pH7 before starting to decrease. However instead of decreasing the enzyme activity was still gradually increasing. This order of testing made it easier to covert the data from the table into a graph.
by
suzy1987 (student)
