LAB REPORT Exercise 11 Enzymes Factors Affecting the Rate of Activity Jim Goetz Lab Section 12 March 13,

2012

Introduction

Living organisms produce enzymes to speed up chemical reactions in their cells (Hershey 2009). Enzymes break down molecules called substrates. Each enzyme has only one substrate in which it breaks down. The location enzymes are produced is in the cells of the body. They affect the rate of almost all chemical reactions that take place in living organisms. Enzyme activity rates are influenced by temperature, pH, and the presence of inhibitors.

Catalase is an enzyme, which is produced by every cell. Its purpose is to break down hydrogen peroxide (H2O2). Hydrogen peroxide is a waste product of cellular activity. It is poisonous to cells. Catalase speeds up the decomposition of hydrogen peroxide into harmless water and oxygen gas (2 H2O2-catalase2 H20 + O2)

Excessive heat will denature enzymes. Denaturization is loss of its structure. This occurs as

ionic and hydrogen bonds break. Salty environments (excess Na+ and Cl-), acidic environments (too much H+), and alkaline environments (too little H+ ) break ionic and hydrogen bonds. This interferes with their electric charges. Heat causes movement within molecules. This disturbes their relatively weak bonds. The pH of a solution will also affect
the charge of acidic and basic amino acid side chains on a protein, affecting the interactions that lead to tertiary and quaternary protein structure. Once denaturated, most proteins will not re-

form their original shape. Enzymes react faster near optimum temperature (Brooker, 2008).
The optimal temperature for the human enzyme is 40 degrees C, while the optimum temperature for enzyme from hot springs prokaryotes is 70 degrees C.

The purpose of this lab was to determine if an inhibitor affects the rate of activity for enzyme catalase. As catalase speeds up the production of oxygen gas; we are able to measure the rate of

enzyme activity, which can be measured as the height of the column of oxygen gas bubbles produced in a test tube. If NaOH or HCL inhibits the action of catalase, then it will slow down the reaction between the catalase enzyme in the meat solution and hydrogen peroxide. .

Hypothesis:

The optimum pH will be 7, and very low pH (pH lower than 7) will denature the enzymes.

Materials and Methods Used in this experiment is distilled water, pH paper, meat solution prepared by lab assistants, hydrogen peroxide, catalase, HCL and NaOH. If lab assistants do not prepare the meat solution, a mortar, pestle and cheese-cloth is needed.

Procedure 11.2 Observe the effects of pH on catalase activity 1. Prepare catalase solution a. Use a mortar and pestle to macerate a marble size portion of fresh, raw ground meat in 10mL of distilled water. b. Filter the solution through cheesecloth into a test tube and add an equal volume of distilled water 2. Obtain 10 test tubes and number them at the top 1-10 3. Obtain stock solutions of distilled water, hydrogen peroxide, buffer of pH 5, buffer of pH 7, buffer of pH 9, 0.1 M HCL, and 0.1 M NaOH. 4. Add distilled water and hydrogen peroxide to each tube as listed in table 11.3. If you are measuring by drops, then 1 mL equals about 20 medium sized drops. Wait 2 min before proceeding to step 5.

5. Add 1 mL of HCL to tubes 4 and 9. Verify that the pH is approximately 3 or lower. 6. Add 1 mL of NaOH to tubes 8 and 10. Verify that the pH is approximately 11 or higher. 7. Add 1 mL of the buffer solutions as indicated in 11.3 8. Use pH paper to measure the values for each solution and record them in table 11.3 9. No catalase is added to tubes 1,3,9 or 10 10. Add catalase to tube 2 according to table 11.3 After adding catalase, swirl the solution gently and immediately record in table 11.4 qualitative changes in the bubbling intensity of oxygen producing on a scale of 0 (no bubbling) to 5 (vigorous bubbling). 11. Repeat step 10 for each remaining solution. 12. Clean work area and materials. Follow instructions for proper disposal of waste solutions containing HCL and NaOH.

Results

Table 11.3

Experimental Conditions To Test The Effect of pH On Catalase Activity Distilled Water 5 mL 4 mL 2 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL

Tube 1 2 3 4 5 6 7 8 9

Buffer H2O2 1 mL, pH 7 1 mL, pH 7 1 mL, pH 7 3 mL 3 mL 1 mL, pH 5 3 mL 1 mL, pH 7 3 mL 1 mL, pH 9 3 mL 3 mL 1 mL, pH 7 3 mL

HCL

NaOH

pH 7 7 7 3 5 7 9 11 3

Catalase Sol 1 mL

1 mL

1 mL 1 mL 1 mL 1 mL 1 mL

1 mL 1 mL

10

1 mL

1 mL, pH 7

3 mL

1 mL

11

Table 11.4 Production of Oxygen Catalase Activity Semi-Qualitiative (0-5)- Oxygen Production 0 1 0 3 2 4 5 5 0 0

Tube 1 2 3 4 5 6 7 8 9 10

The result of this experiment was there was no breakdown in any solutions without the catalase to break down the molecules. Without the proper enzyme for a reaction to occur, no reaction will occur.

The graph of the results show that as the pH increases, so too does the reaction, evidenced by 02 production.

The independent variables in this lab were the addition of HCL and NaOH and the dependent variable is the amount of oxygen gas produced (measured as the height of gas bubbles). The negative control is distilled H2O + H2O2 while the positive control is the H202 + catalase (neutral).

Discussion It appears that the lower the pH, the lower the reaction, which confirms my hypothesis. When the pH was 3 or 5, the reaction yielded minimal results. When the pH was over 7 (neutral) the

reaction was much greater. The reaction at a pH of 7 was the median result. This shows that anything acidic will denature enzymes. The more alkaline the solution the less denaturization of the enzyme.

Each and every enzyme is characterized by an optimum pH. At a specific pH level, a specific enzyme catalyzes the reaction at the fastest rate than in any other pH level. An example of this; the enzyme pepsin (a protease enzyme) that catalyzes proteins is most active at an acidic pH, whereas the enzyme trypsin (another protease enzyme) performs best at a slightly alkaline pH. Catalases optimum pH is 7. The results obtained showed catalase reacting to a pH much higher than 7. This may have been due to a misreading of the pH strips or the fact that the results of the semiqualitative results of the oxygen production is subjective (most likely the case) The optimum pH of an enzyme is different from that of another enzyme. pH is defined as the measurement for the acidic or alkaline nature of a solution. More specifically, pH indicates the concentration of dissolved hydrogen ions (H+) in the solution. An increase or decrease in the pH changes the ion concentration in the solution. These ions alter the structure of the enzymes and the substrate may form additional bonds or breakage of already existing bonds. The chemical makeup of the enzyme and substrate change, while the active site of an enzyme is changed. After this the substrate can no longer identify the enzyme.

References Brooker, Robert J, et al. Biology. New York: McGraw-Hill, 2008. Hershey, James, et al. Biology 110 Laboratory Textbook. New York: Pearson, 2009.