Sheikh, Sehar Effects of pH on Catalase Enzyme Activity INTRODUCTION To obtain a healthy lifestyle, various numbers of chemical reactions are

occurring in our body with the help of particular enzyme to designate a certain task. An enzyme is a protein well-known for speeding up and controlling the reaction. The presence of an enzyme has allowed the completion of chemical reactions even under normal body temperature of 37 degrees Celsius. Catalase found in beef liver serves to protect the cell from the toxic effects of hydrogen peroxide (Pape-Lindstrom 2011). The functioning of the enzyme is determined by the shape of the protein and many enzymes function by lowering the activation energy of reactions (M.J. Farabee). In this reaction, substrate of hydrogen peroxide will break its bond with the help of catalase into oxygen and water. 2 H2O2
catalase

2 H2O + O2

The hypothesis stated that enzymes being sensitive to pH would work best under neutral condition of pH 6; however, most acidic buffer will generate less oxygen than most basic buffer. In most acidic solution, the enzyme would denature and change its structure because it wont be able to catalyze the reaction due to excess hydroxide ions. METHODS The experiment was set up by collecting essential instruments such as graduated cylinder, flask, tub, little beakers, and rubber bung equipped with delivery tube and syringe. The buffer solution was obtained from the counter-top ranging different concentrations. We have used pH 2, pH 6, pH 10, and pH 12. The little beakers were used for measurements. We added 10mL substrate of hydrogen peroxide to the flask, 2 mL of pH solution, measured with pH paper for confirmation, and shut the flasks neck with rubber bung. On the other hand, the tub was filled with water and graduated cylinder was tilted carefully in regards to allowing water to be filled without giving any access to the oxygen. The graduated cylinder was placed upside down inside of the tub. The other end of the delivery tube was placed into graduated cylinder. The syringe was used to insert 2mL of catalase enzyme into the flask while being attached to the rubber bung tightly. The flask was consistently swirled in circular motion with one hand to keep a steady reaction. The timing was recorded with 1 min interval for up to 5 minutes as we measured the oxygen production in the graduated cylinder. The trials were repeated twice with all buffer solutions; however, second trial data was only used in this experiment for interpretation and results. The control was with distilled water instead of buffer solution. The independent variable in the experiment was different pH levels of buffer solutions while dependent variable was Oxygen production with respect to time. 1

Sheikh, Sehar

Demonstration of Experimental Set-Up:

RESULTS Table: Production of O2 Time 1 min 2 min 3 min 4 min 5 min pH = 2 1 mL 1 mL 2 mL 2 mL 2 mL pH = 6 66 mL 98 mL 104 mL 104 mL 104 mL pH = 10 56 mL 98 mL 102 mL 102 mL 102 mL pH = 12 36 mL 76 mL 102 mL 104 mL 104 mL Control 60 mL 98 mL 98 mL 98 mL 98 mL

Sheikh, Sehar Graph: pH effect on Enzyme Activity

pH effect on Enzyme Activity
Oxygen Production (mL) 120 100 80 60 40 20 0 1 min 2 min 3 min 4 min 5 min Time (minutes) pH = 2 pH = 6 pH = 10 pH = 12 Control

The enzyme catalase was severely affected by the most acidic pH of 2 that eventually denatured the protein quite rapidly. Comparing pH 6 to pH 12 demonstrated a definite trend leading more oxygen production with less concentrated buffer solution. DISCUSSION/CONCLUSIONS The prediction that enzyme activity would work best under neutral pH 6 was supported by the data. Also, the assumption about pH 2 (most acidic) buffer changing catalases shape to not complete the chemical reaction came to be true because the substrate of hydrogen peroxide could not attached to the binding site as the protein function is determined by the structure. The pH ranging from 6 12 in presence of an enzyme allowed the substrate to bind to the active site to produce oxygen from 36mL to 104 mL while hydrogen peroxide bonds were being broken down into water and oxygen. The data interpreted production of O2 in 5 minute span while the reaction originally stopped in 3 minutes. The results indicated production of O2 in 3 minutes: pH 6: 104 mL, pH 10: 102 mL, pH 12: 104 mL, and control produced 108 mL of oxygen. Trial 1 and Trial 2 were completely different in O2 production that might be influenced by inaccurate measurements. The enzyme was able to catalyze the reaction much faster for pH 6, 10, and control, if compared with pH 12. The control with distilled water, pH level of 6, and produced 98 mL of O2 in 2 minutes indicated the fact that the reaction was catalyzed 1 minute earlier than all of the buffer solutions. In 1 minute, the most basic buffer solution took more time to generate oxygen but it increased rapidly after that when catalase lowered its activation energy. To confirm pH level, pH paper was used to double-check the performance of the experiment at the end of each reaction. All of the pH level matched with indicated buffer except pH 12 that resulted into pH 10, making it to be 100 times more acidic than 3

Sheikh, Sehar being the most basic buffer. The pH 12 buffer was required to have more H+ concentration than OH- concentration that could completely change our results and approach to the experiment. However, pH 12 and pH 6 produced about the same volume of oxygen but it took pH 12 three minutes to reach maximum production and two minutes for pH 2. The graph showed similar trend where one can observe the relationship between pHs that the catalase reacted effectively within three minutes. An additional question that might be investigated is changing the enzyme concentrations to see if the results can vary in O2 production and how pH would relate to enzymatic activities. Enzymes exhibit saturation kinetics; as the relative concentration of substrate increase, the rate of the reaction also increases (Pg. 11, Orsay). Also, changing the volume of substrate concentration can be applied to this experiment for better results because the reaction might have stopped early as the hydrogen peroxide was used up completely. In Conclusion, an enzyme wont be able survive in high temperatures or most acidic solutions because they are adapted to certain pH range to perform a chemical reaction.

LITERATURE CITED o Farabee, M.J. "REACTIONS & ENZYMES." Estrella Mountain Community College. 18 May 2010. Web. 05 Feb. 2012. <http://www.emc.maricopa.edu/faculty/farabee/biobk/biobookenzym.html>. o o . Lindstrom, Pape. Biology 222 Lab Manual. Everett Community College. 20 December, 2011. Print

Orsay, Jonathan. ExamKrackers MCAT Biology. New Jersey: Osote Pub., 2007. Print.