PRACTICAL 8 Title Objective Introduction : Investigation on the influence of substrate concentration to the rate of the catalase reaction.

: To investigate how substrate concentration influence the rate of the catalase reaction. : Enzymes are usually specific for particular reactions and have effects on certain reactant only. This experiment involves liver as the source of catalase where it will increase the rate of biochemical reaction (in this case, the formation of oxygen gas from hydrogen peroxide). The reaction produces oxygen and forms foam if detergent is added. Formation of foam will be a measure of the enzymes-driven reaction.

Research Question : How the substrate concentrations influence the rate of the catalase reaction? Hypothesis : The higher substrate concentration, the higher catalase reaction until the reaction is not change. In this experiment, the reaction becomes constant at the certain time because the catalase is the limiting reagent. : a) Constant : the cubes size of liver (the amount of catalase), temperature b) Independent: the concentration of hydrogen peroxide (6%, 4%, 2%, 1%, 0.5% and distilled water) c) Dependent : the volume of the foam produced (measure of the enzyme activity)

Variables

Apparatus Materials Method

: Stop watch, 1 x 5 mL or 10mL measuring cylinder, sharp knife, forceps, 1 x 100 mL measuring cylinder, 2 teat pipettes. : Solutions of hydrogen peroxide (6%, 4%, 2%, 1%, 0.5%), detergent, fresh liver, distilled water. :

1) 6 cubes of liver each approx was cut into 1cm x 1cm x 1cm. 2) The small measuring cylinder and teat pipette were used to measure 4mL of 6% hydrogen peroxide with two drops of detergent into a 100mL measuring cylinder and mix by swirled them. 3) One cube of liver was taken and placed in the measuring cylinder by using the forceps.

4) This procedure was repeated with the other 4 solutions of hydrogen peroxide and also for distilled water. Make sure your glassware was rinsed carefully between procedures. 5) Either this procedure was repeated twice or the results were obtained from two other groups. Data Collection: Temperature : 30.5oC Initial volume : 5 mL The volume of the solutions (hydrogen peroxide + detergent + foam) reaches The concentration of hydrogen peroxide (%) Distilled water 0.5 1.0 2.0 4.0 6.0 Data Processing We define the volume of the foam reaches by using this formula, The volume of the solutions (hydrogen peroxide + detergent + foam) 5 (the volume of 4 mL hydrogen peroxide + 1 mL detergent), The volume of the foam reaches Solutions of hydrogen peroxide (%) Distilled water 0.5 1.0 2.0 4.0 6.0 The volume of the foam reaches (mL) 1 0.0 4.0 12.0 15.0 29.0 35.0 2 0.0 6.0 12.0 15.0 29.0 33.0 3 0.0 4.0 11.0 17.0 31.0 35.0 The volume of solution reaches, X (0.5ml) 1 2 3 5.0 5.0 5.0 9.0 11.0 9.0 17.0 17.0 16.0 20.0 20.0 22.0 34.0 34.0 36.0 40.0 38.0 40.0

From this, we can calculate the average of total volume of the foam using this formula, The average of total volume of foam = 1 + 2 + 3 readings 3 The table of average of total volume of the foam Solutions of hydrogen peroxide (%) Distilled water 0.5 1.0 2.0 4.0 6.0 The average of volume of the foam (mL) 0 4.667 11.667 15.667 29.667 34.333

We can calculate the reaction rate of the foam using this formula, Reaction rate/ mL s-1 = Average total volume of foam 30s The table of the reaction rate of the foam Solutions of hydrogen peroxide (%) Distilled water 0.5 1.0 2.0 4.0 6.0 Reaction rate (mL s-1) 0 0.156 0.389 0.522 0.989 1.144

From this table, the graph of reaction rate of the foam can draw,

T h e G r a p h o f T h e R e a c tio n R a te o f T h e F
1 .4 1 .2 1 Reaction Rate (mL/s) 0 .8 0 .6 0 .4 0 .2 0

0 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6
S o l u ti o n s o f H y d r o g e n P e r o x i d e (% )

Conclusion: From the results, we know that the hypothesis is true and accepted. The results show that if the concentration of the hydrogen peroxide is higher, then the volume of foam that represents the reaction rate is higher until it reaches a constant value. Discussion : The liver contains catalase enzymes that increase the rate of reaction of hydrogen peroxide into water and oxygen. Hydrogen peroxide is a very reactive chemical. Catalase breaks the substance of hydrogen peroxide into two substances that are hydrogen peroxide and oxygen. The presence of hydrogen peroxide in cells could cause toxic environment in cells. Thus, it must be removed. The hydrogen peroxide is a by-product of cellular reactions. Catalase which is found in most tissues from living organism helps removing the hydrogen peroxide as it increase the reaction below. 2 H2O2

2H2O + O2

From the graph, we could derive that with the increase of the reactants (H2O2) concentration, the rates of reaction also increase. (we can take the enzymes factor at constant)

Evaluation: Limitation The volume of foam produced may not be accurate as oxygen tends to be released to air. Recommendation The experiment could be conducted over and over for many times. When we carry out the experiment for many times, we will get more data and average of the data must be more accurate. The apparatus must be cleaned and dried throughly between the procedures in the experiments.

The apparatus used may contain left over during procedures between the experiments. This will cause excess solution or enzymes in the experiments. The actual sizes of livers in each experiment are not accurately same. This will cause some disruptions in the reaction as enzymes play important roles.

We must cut the liver into cubes which are at the same size. To get same size of each cubes, cut the livers carefully.