Lab Report: Enzyme Activity

Lab Report: Enzyme Activity

Introduction

Background

Chemical reaction in general can be accelerated in a number of ways. Two important ways are by an increase in temperature up to the optimum and by the use of catalysts. A catalyst is a substance that increases the rate of a reaction without being used up itself. Enzymes are protein molecules that function as catalysts and speed up reactions in living organisms without the need to raise the temperature of the reaction
Enzymes are usually specific for particular reactions and work best when conditions such as pH, temperature and ion concentration are most suitable. If these conditions are inappropriate for that enzyme, the reactions will proceed at a slower rate or not at all.
Hydrogen peroxide (H₂O₂) is a very reactive chemical that is formed as a by-product in cellular reactions. It is highly toxic and must be removed or it will disrupt chemical reactions in the cell. An enzyme, catalase, which is found in most tissue from living organisms, breaks the substrate (H₂O₂) down into two harmless substances, water and oxygen, according to this equation:

2H₂O₂ à 2H₂O + O₂

In this experiment liver will be used as a source of catalase.
The oxygen is given off as a gas and, if detergent is added to the substrate, foam is formed.
The volume of the foam produced in a given time can be used as a measure of the enzyme activity.

Aim

Our aim in this experiment is to use this reaction to investigate how substrate concentration may influence the rate of the above reaction.

Equipment

Materials required

Solutions of hydrogen peroxide (at 16%, 8%, 4% and 2%)
detergent
stop watch or clock
1 x 5 ml measuring cylinder
Sharp knife or razor blade
forceps
fresh liver
1x 100 ml measuring cylinder
2 x teat pipettes
distilled water

Experiment

Method

1. We cut 5 cubes of liver each approximately 1cm³

2. We then used the small measuring cylinder and teat pipette to measure 4 ml of 2% hydrogen peroxide with to drops of detergent into a 100 ml measuring cylinder and swirled it for it to mix.

3. Using the forceps we took one cube of liver and placed it in the measuring cylinder. After 30 seconds, we recorded the volume the foam reached in the cylinder and recorded our data in a table seen below.

4. After that we repeated this procedure with the other 3 solutions of hydrogen peroxide and did the same thing with distilled water. We also made sure we rinsed our glassware carefully between procedures.

5. Then we obtained results from five other groups and entered them in the table below.

The independent variable during this experiment was the substrate concentration which we used. The dependent variable was the amount of foam formed.

Results:

	Foam/ +- 1ml
Group nr. Sub. Con. %	1	2	3	4	5
2	16	22	16	20	14
4	23	30	35	35	24
8	50	70	77	40	60
16	105	35	130	80	130
0	0	0	0	0	0

Graph A. The dynamic change of enzyme activity as a result of increased substrate concentration in different groups:

*I was in group nr 3

Graph B. The dynamic change of enzyme activity as a result of increased substrate concentration using the average of all groups:

Calculations of average:
For the substrate concentration of 0% (H₂O) = 0+0+0+0+0 = 0
For the substrate concentration of 2% H₂O₂ = 16+22+16+20+14/5 = 17.6
For the substrate concentration of 4% H₂O₂ = 23+30+35+35+24/5 = 29.4
For the substrate concentration of 8% H₂O₂ = 50+70+77+40+60/5 = 59.6
For the substrate concentration of 16% H₂O₂ = 105+35+130+80+130/5 = 96

*All the results above are given in ml.

Conclusion and Evaluation

Our aim in this experiment has been to investigate how substrate concentration affects the activity of certain enzymes, in this case, catalase. From the results of our experiment, graph A and graph B, we can see clearly that the activity of the reaction taken place increases as we add a more concentrated substrate, H₂O₂. When we add water as a substrate, containing no H₂O₂, no reaction is taken place at all. From this we can draw the conclusion that the more concentrated the substrate is, the more reaction will take place with the help of the enzyme catalase, producing more water and oxygen (foam).
If we compare our results to the IB-study guide, we can observe that the curve of our graph is quite inaccurate. On the next page is an example of a more correct graph displaying the enzyme activity of increasingly concentrated substrates:

Here we can observe that the difference between our results and this example of a correct graph can be summed up into two main points:

· The rate of reaction increases drastically initially, because of the rapid increase of substrate concentration more reaction takes place and more active sites are being occupied.

· At one point at the latter part of the graph all the active sites in the enzymes are occupied. This makes it impossible for an increase of reactions as no more substrate concentration can be broken down without additional enzymes.

The problems and difficulties which caused this inaccuracy in our results might have been caused by several reasons, but the three most obvious limitations of this lab can be summed up in three points:

· The liver was supposed to be cut into pieces of the size of one cubic centimeter each and it is extremely difficult if not impossible to do this manually. Different parts of the liver can also contain different amounts of catalase which again makes this highly inaccurate.

· Secondly, the amount of detergent needed being referred to as “two drops” makes the lab even more imprecise. Two drops is not a static volume and is therefore not a precise way of describing an amount of any substance. The amount of detergent increases the amount of foam that is produced so it is very vital during this experiment and has a large effect on the result.

· Finally, the volume of the foam, which has a critical impact on the result, is very imprecise. The density of the foam can differ, and thus the volume, which gives us an incorrect appreciation of the reactions taken place.

There are several things we could do to improve this experiment to gain a more accurate result. The catalase can be measured more accurately if the substance is purified in a chemical form, the amount of detergent could have been given with a more exact volume of measurement and finally the reactions taken place could have been measured in a different way than by measuring the amount of foam produced.

Summary

During this lab we have investigated how substrate concentration affects the activity of enzymes, in this case catalase. We did this by creating a reaction of the breaking down of a substrate and then recording how much the volume of the product of the reaction differed between different concentrations of the substrate. The result we got gave us a clear picture of how increased substrate concentration created a dynamic change of the reactions taking place. The result was however very imprecise for several reasons but the one thing which we could still conclude from our results was that the dynamic change that takes place in amounts of reaction when a different substrate concentration is added. When we compared this to our literary source we could further conclude that the enzyme activity or the rate of reactions which take place, increase until a certain point when all active sites of the enzymes are occupied, and the rate of the reactions can no longer increase.