Enzyme Experiment Design

by nathan_chang (student)

Enzyme Activity

Introduction

Enzymes are globular protein, which is made up by chains of amino acids, that acts as a catalyst for biochemical reaction. Enzyme will be able to work as a catalyst when a substrate (a reactant in a biochemical reaction) is present and bind with the active site on enzyme, which is the area on the surface of an enzyme.

In this experiment, the enzyme is catalase and the substrate is hydrogen peroxide. Using catalase hydrogen peroxide will break down into water and oxygen, which can be shown by the chemical equation: 2H2O2 2H2O + O2

Also, the rate of enzyme activity can be affected by different factors including temperature, pH level and substrate concentration.

Aim

In this experiment, I will be investigating how the change in hydrogen peroxide concentration can affect the rate of enzyme activity. In order to do this I will measure the amount of oxygen produced in a certain period for different concentration of hydrogen peroxide. My aim is to find the maximum rate of reaction at which the enzyme is working at its greatest efficiency when increase in concentration will result in no increase in enzyme activity.

Research Question

What is the effect of substrate (hydrogen peroxide) concentration on the rate of reaction between hydrogen peroxide and catalase?

Hypothesis

I believe that as the substrate concentration (hydrogen peroxide concentration) increases that rate of reaction will also increases. However, as it reaches the maximum substrate concentration, when all active site are full and working at maximum efficiency. As a result, any increase in substrate concentration beyond the maximum point will have no added effect as there are no extra active sites that can be used.

Variables

Dependent Variable

※Volume of oxygen gas released in 10 ...

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Research Question

What is the effect of substrate (hydrogen peroxide) concentration on the rate of reaction between hydrogen peroxide and catalase?

Hypothesis

Variables

Dependent Variable

※Volume of oxygen gas released in 10 seconds

Independent Variable

※Hydrogen peroxide concentration: 0%, 1%, 5%, 10%, 15%, 20%

Controlled Variable

※The temperature of the hydrogen peroxide and catalase is kept at room temperature which is about 23.5℃ when I measured it. By keeping the temperature constant can prevent it acting as a contributing factor that will affect the reliability of my results.

※Throughout the experiment, I will only be using 1% catalase solution.

※The volume of catalase (3ml) and hydrogen peroxide(2ml) is to be keep constant 3ml catalase, by using measuring cylinders.

※Time – I will only measure the amount of oxygen produced in the first 10 seconds of the reaction.

Uncontrolled Variable

※pH level of hydrogen peroxide concentration:

source: http://h2o2.com/faqs/FaqDetail.aspx?fId=26

As shown from the table above, different concentration of hydrogen peroxide has different pH level. It can’t be possible for me to keep the pH level of hydrogen peroxide constant while changing its concentration; therefore even though pH level may affect the rate of enzyme activity, I cannot control the pH level as the change in pH level may also change the concentration of hydrogen peroxide.

Apparatus

1% catalase – 3ml x 25 (75ml in total)
hydrogen peroxide (1%, 5%, 10%, 15%, 20%) – 2ml x 5 for each concentration: 10ml in total
2 measuring cylinders (10ml) and 1 pipette
2 beakers (50ml) and 1 conical flask (50ml)
1 glass syringe (100cm3)
10 test tubes and a test tube rack
1 ring stand
1 delivery tube and 1 stopper and blu tack
1 stopwatch

safety goggle and plastic gloves

Risk Assessment

This procedure uses hydrogen peroxide. Wear safety goggle and plastic gloves.

Method

Gather all needed apparatus and set it up like the picture beside. Rinse all equipment and place test tube in the test tube rack. Attach the glass syringe firmly on to the clamp of the ring stand and connect the delivery tube to the tip of the glass syringe and connect the other side of the delivery tube with a stopper to the conical flask, use blu tack to seal the junctures.
Prepare 15ml of 1% catalase and 10ml of 20% hydrogen peroxide.
Use measuring cylinder and pipette to get 3ml of 1% catalase and 2ml of 20% hydrogen peroxide. Check the measurements at eye level to ensure that the measurements are exact. And pour each solution into a test tube. Repeat this for 5 times.
Pour the catalase into the conical flask. Pour a test tube that contains 2ml of 20% hydrogen peroxide into the conical flask.
Have the stopwatch ready and quickly cover the conical flask with the stopper that is attached to the delivery tube and immediately at timing with the stopwatch
When the time almost reaches to 10 seconds keep your eyes close to the plunger for the amount of oxygen produced. Pull the stopper out from the flask and record the result down.
Push the plunger back and rinse conical flask
Repeat steps 2 to 9 for five times for each different hydrogen peroxide concentration.