The Effect of pH on Enzyme Activity

The Effect of pH on Enzyme Activity Abstract This experiment was performed in order to find a better understanding of whether or not pH will affect the reaction rate of peroxidase. To test this, we had a 20 second time frame to mix together substances with different pH’s in order to test whether or not that specific pH was ideal for the tube. The results were as predicted, the pH increased the reaction rate if the solution was more neutral, whereas the pH that was more acidic or basic caused the reaction rate to decrease almost completely. The tubes that were given a more acidic or basic pH barely had a low absorbance rate, whereas, the tubes given the more neutral pH had a high absorbance rate. My hypothesis, changing the pH will change the rate of the reaction by either increasing or decreasing it, was accepted. Introduction Peroxidase is a type of enzyme that breaks down toxic hydrogen peroxide into a less harmful substance, that is easily found in turnips, horseradish roots, and animal tissues. The purpose of this experiment was to retain a deeper understanding of how the pH will or will not alter the enzyme activity of peroxidase. In order to measure the absorbance rate, a spectrophotometer is used. A spectrophotometer measures two different things, either the amount of light reflected from something or the amount of light that is absorbed (Kevin Vo, 2015). In this experiment, the amount of light being absorbed in the spectrophotometer is being measured. As it is highly known that when the pH will change the shape of an enzyme (McGraw-Hill 112). Temperature, pH, and concentration gradients are key factors to the shape of an enzyme. Therefore, when one of those are altered, the shape of the enzyme will change and cause the rate of the reaction increase or decrease. The peroxidase is used to functioning at the pH of 5 but testing the other buffers would help us understand if the peroxidase would work as well. The solutions with a more neutral pH will have the highest rate of reaction, whereas the solutions with a more acidic or basic pH will have little to no rate of reaction. Changing the pH of a solution will change the rate of the reaction. Materials and Methods Performed in a Laboratory Setting Each of the nine tubes were previously assigned a certain amount of buffer (pH), hydrogen peroxide, peroxidase, and guaiacol (dye). For example, test tube 1 got 5 milliliters of buffer, 2 milliliters of H2O2, no peroxidase, and 1 milliliter of guaiacol. Each tube was filled with the desired substance with a separate pipette for each one. The buffers ranged in pH from 3 to 9. In a 20 second time frame, one person was to mix two of the tubes together by pouring them back and forth twice. Then, the same person used an eye dropper to place some of the mixture into a cuvette. That person handed the cuvette to a different member of the group to wipe down the outside of it. She would then place the cuvette in the spectrophotometer. The number the spectrophotometer says at the point of 20 seconds was then drawn into the table and data was recorded at 20 second intervals. Another group member took down the data as the timer called out each 20 second mark for 2 minutes. The cuvette was then removed from the machine and each member visually noted the color change. Tubes mixed together were 2 and 3, 4 and 5, 6 and 7, and 8 and 9. Results After the experiment, my lab partners and I had gone through the data and decided what really happened. The mixture of tubes 2 and 3 had a total pH of 3, meaning, the pH was too acidic for the peroxidase to work properly. The 4 and 5 mixture had the pH level of 5 which is where the peroxidase is used to functioning. This mixture worked very well and the absorbance rate was quick; the absorbance rate was already at 0.068 by the 20 second mark (Figure 1). The mixture of 6 and 7 had the most optimal level of pH of 7. The almost basic level of pH caused the reaction to go fastest and reach to the highest rate of absorbance out of the four tube mixtures. Finally, the 8 and 9 mixture was not good. The reaction was just barely there. Discussion I accept my hypothesis because the data showed that the rate of reaction from each tube were all fairly different due to the differed values of buffer. None of the results were unexpected because I assumed that the peroxidase would work best with a neutral pH, even though all enzymes are affected differently by pH . The peroxidase did not function because the solution was either too basic or acidic. Peroxidase is found in all different organisms such as humans, plants, and animals (NKU). In plants, peroxidase can be found in the roots of horseradish and turnips. In humans, peroxidase can be found in thyroid glands and secretions such as breast milk, tears, and saliva (George Hajishengallis, 2015). This lab could have been done differently by having a wider time frame than just twenty seconds. I feel that we were rushed to beat the twenty second mark causing us to potentially make mistakes that we could not have caught in that short amount of time. I also think the lab could. Sttndnnf o,..,1Jon otтnal Pf'�IIOAmounts .. ' -I.... J41 -,.."' -twllr.litl.1 ..... .,. Temper1ture Effecu оп Peroкld1se � о, t. Ol f J"аа " " ш lO 40 .... IWМЬ,кandt,I -J.,Ь.,,H1J -t..и<11&S -,....... 'f -n.ь...1-1• Stвrd.ardDe\11.at'IDn ....� р of �emperatureEffectsero:c1da.R!' ,--------:----:" , ____J 16 80 r .. ____, :оо