INTRODUCTION An enzyme is a checmical molecule made up of proteins that serves as a catalyst (speeds up chemical reactions). http://www.s-cool.co.

uk/gcse/biology/enzymes/revise-it/enzymes Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. The reaction that catalase speeds up is the one for the decomposition of living tissue (2 H2O2 2 H2O + O2) http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Catalase.html In this lab, I am attempting to answer a multitude of questions dealing with catalase concentration, namely how different factors affect its reaction rate. There are four different questions that I am attempting to answer in this lab; how does the concentration of catalse affect the reaction, how does the ceoncentration of hydrogen peroxide affect the reaction, how does heat affect the reaction, and how does pH affect the reaction. In order to determine how the concentration of catalse affects the reaction, I am going to take 5 vials of 100units/ml catalase and and dilute it into five different concentrations. Then, I will put filter paper into each concentration three times, and time how long the paper takes to rise to the surface of a vial of 40 ml 3% hydrogen peroxide. For the second question I will take 5 vials of hydrogen peroxide and dilute each on into a different concentration. I will then put filter paper into each concentration three times, and time how long the paper takes to rise to the surface of the vial. For the third question, I will put water of varying temperatures into four different cups, and put a test tube of catalase into each one. Then, I will put filter paper into each of the test tubes three times and time how long it takes to rise to the top of a 40ml beaker of 3% concentration hydrogen peroxide. For the fourth question, I will set up five vials with varying pH and 5 ml catalase, and put filter paper into each of these vials three times, then put the paper into 40ml 1% H2O2 and time how long it takes for the paper to reach the surface of the beaker. HYPOTHESES My hypothesis is that a higher catalase concentration will result in a faster reaction time, a higher 3% hydrogen peroxide concentration will result in a faster reaction time, and temperatures closes to body temperature will have the fastest reaction time. PROCEDURE
GENERAL DIRECTIONS:

The assay system used in this lab consists of a filter paper disc which is coated with the enzyme and then dropped into a vial of substrate (hydrogen peroxide). As the catalyst breaks down the hydrogen peroxide into water and oxygen gas, the bubbles of oxygen collect underneath the filter and make it rise to the surface of the hydrogen peroxide. The time it takes for the filter to rise is an indication of the rate of enzyme activity.

PLAN:

Each group will investigate and report on two of the following questions. Suggestions for each question are given below. EVERY STUDENT IS RESPONSIBLE FOR RECORDING THE RESULTS OF ALL FOUR EXPERIMENTS in his/her lab report. 1. What is the effect of enzyme concentration on enzyme activity?

2. What is the effect of substrate concentration on enzyme activity? 3. What is the effect of pH on enzyme activity? 4. What is the effect of temperature on enzyme activity?
1. What is the effect of enzyme concentration on enzyme activity?

Set up five vials containing 40 ml of 3% hydrogen peroxide each. Measure and record the depth of the hydrogen peroxide in the vials. Dilute the enzyme as follows. Make each dilution in a separate cup. 100 units/ml = 20 ml 100 units/ml 80 units/ml = 12 ml 100 units/ml + 3 ml cold dH2O 50 units/ml = 10 ml 100 units/ml + 10 ml cold dH2O 20 units/ml = 3 ml 100 units/ml + 12 ml cold dH2O 0 units/ml = 20 ml cold dH2O

Using forceps, dip a filter into the enzyme solution at 100 units/ml, then remove it and drain it on a paper towel. Drop the disc into the vial of hydrogen peroxide labeled 100 units/ml and time how long it takes the filter to rise to the surface. Repeat this procedure for each of the other enzyme dilutions, and be sure to use a FRESH vial of substrate for each filter (WHY?) Record your results in the appropriate data chart.
2. What is the effect of substrate concentration on enzyme activity?

Obtain 1 vial of catalase at 100 units/ml Dilute the substrate (hydrogen peroxide) as described below. Each dilution should be made in a separate labeled vial. Measure and record the depth of the hydrogen peroxide. 3.0% H2O2: 40 ml 3% H2O2 1.5% H2O2: 20 ml 3% H2O2 + 20 ml distilled water 0.75% H2O2: 10 ml 3% H2O2 + 30 ml distilled water 0.38% H2O2: 5 ml 3% H2O2 + 35 ml distilled water 0.0% H2O2 : 40 ml distilled water

Dip a filter into the catalase, drain on a paper towel and then drop the filter into the 3% H2O2. Time how long it takes the filter to rise to the top. Repeat this procedure for each of the substrate dilutions. Record your results in the appropriate data chart.

3. What is the effect of pH on enzyme activity?

Obtain 1 vial of 40 ml 1% H2O2. Measure and record the depth of the hydrogen peroxide. Label 5 small vials as follows: pH3, pH5, pH7, pH9, pH11 and dilute catalase into the appropriate vial as directed below: pH 3: 5 mL catalase + 5 mL pH 3 Buffer pH 5: 5 mL catalase + 5 mL pH 5 Buffer pH 7: 5 mL catalase + 5 mL pH 7 Buffer

pH 9: 5 mL catalase + 5 mL pH 9 Buffer pH 11: 5 mL catalase + 5 mL pH 11 Buffer Dip a filter into the catalase at pH 3, drain on a paper towel and drop it into the 1% H2O2. Time how long it takes the filter to rise to the top. Remove the filter and repeat this procedure for each of the pH's. Record your results in the appropriate data chart.

4. What is the effect of temperature on enzyme activity?

Set up an ice bath (0oC), a room temp water bath, a 37 oC bath and a boiling water bath Place 5 ml of catalase at 100 units/ml in each of 4 test tubes. Place 1 test tube in each of the water baths. Place 40 ml 1% H2O2 in each of 4 vials. Measure and record the depth of the H2O2. Place 1 vial in the 0oC bath and leave 3 at room temperature. This is necessary because heat will destroy the hydrogen peroxide. Allow the catalase and substrate to incubate at each temperature for about 5 minutes, then test the reaction time at each temperature by dipping a filter into enzyme at that temperature, draining it, and then dropping it into substrate at the same temp. Use the second room temp. vial of hydrogen peroxide for the boiled catalase. DO NOT BOIL HYDROGEN PEROXIDE!!! Time how long it takes the filter to rise at each temperature. Record your results in the appropriate data chart.

DATA
EXPERIMENT 1 Height of Vials for filter papers catalase % to 40 mL H2O2 Vial 1 (100 Catalase 31.2 mm 8.807 s 8.986 s 10.440 s 81.2 mm 0.0% >106 s >106 s >106 s Vial 4 (Ice Bath) 32.2 mm 20.48 s 20.26 s 17.96 s Vial 2 (80 Catalase) 31.1 mm 13.351 s 11.171 s 14.650 s 82.1 mm 0.38% 53.23 s 45.73 s 40.95 s Vial 3 (Room Temp) 31.6 mm 21.88 s 19.03 s 20.13 s Vial 3 (50 Catalase) 32.1 mm 12.946 s 11.323s 15.245 s 81.8 mm 0.75% 27.28 s 28.73 s 28.11 s Vial 2 (Body Temp) 31.8 mm 33.15 s 39.07 s 43.07 s Vial 4 (20 Catalase) 31.6 mm 36.679 s 61.394 s 111.624 s 80.9 mm 1.5% 17.40 s 13.31 s 14.20 s Vial 1 (Boiling) 31.4 mm >86 s >86 s >86 s Vial 5 (0 Catalase) 31.1 mm >224 s >224 s >224 s 81.9 mm 3.0% 12.21 s 9.18 s 8.86 s

EXPERIMENT 2 Height of Vials for filter papers 100% catalase to % H2O2

EXPERIMENT 4 Height of Vials for different temperatures

1. What is the effect of enzyme concentration on enzyme activity?

2. What is the effect of substrate concentration on enzyme activity?

3. What is the effect of pH on enzyme activity?

4. What is the effect of temperature on enzyme activity?