An investigation into the effect of substrate concentration on the activity of the enzyme catalase in processed liver.

By Steven Duong

Background

Catalase behaves as a catalyst for the conversion of hydrogen peroxide into water and oxygen. Catalase is an example of a particularly efficient enzyme. Catalase has one of the highest turnover numbers for all known enzymes (40,000,000 molecules/second). This high rate shows an importance for the enzymes capability for detoxifying hydrogen peroxide and preventing the formation of carbon dioxide bubbles in the blood.

Advantages of hydrogen peroxide:

Powerful - H2O2 is one of the most powerful oxidizers known -- stronger than chlorine, chlorine dioxide, and potassium permanganate. And through catalysis, H2O2 can be converted into hydroxyl radicals.

Safe - Despite its power, H2O2 is a natural metabolite of many organisms, which decompose the H2O2 they produce into oxygen and water. H2O2 is also formed by the action of sunlight on water -- a natural purification system for our environment.

Versatile - The fact that H2O2 is used for seemingly converse applications proves its versatility. For example, it can inhibit microbial and encourage microbial growth. Similarly, it can treat both easy-to-oxidize pollutants and difficult to oxidize pollutants.

Selective - The reason why H2O2 can be used for such diverse applications is the different ways in which its power can be directed. By simply adjusting the conditions of the reaction (e.g., pH, temperature, dose, reaction time, and/or catalyst addition), H2O2 can often be made to oxidize one pollutant over another, or even to favour different oxidation products from the same pollutant.

Widely Used - Since it was first commercialised in the 1800's, H2O2 production has now grown to over a billion pounds per year (as 100%). In addition to pollution control, H2O2 is used to bleach textiles and paper products, and to manufacture or process foods, minerals, petrochemicals, and consumer products (detergents).

Enzymes such as Catalase are protein molecules, which are found in living cells. They are used to speed up specific reaction within the cell. They are all very specific as each enzyme just performs one particular reaction. Catalase is an enzyme found in food such as potato and liver. It is used for removing Hydrogen Peroxide from cells. Hydrogen Peroxide is the poisonous by-product of metabolism. Catalase speeds up the decomposition of Hydrogen Peroxide into water and oxygen:

Hydrogen peroxide catalase water + oxygen

(2H2O2) (2H2O) + (O2)

Enzymes are globular protein molecules known commonly as Biological Catalysts. The definition of a catalyst is a substance that can be used to speed up the rate of a chemical reaction while remaining unaffected and unchanged after the reaction. The enzyme’s ability to act relies strongly upon the specific shape of the protein molecule, which is coiled into a precise three-dimensional shape. This specific shape is the cleft or depression, which other molecules (substrates) fit into perfectly; this part of the enzyme, is called the active site. The tertiary folding of the polypeptides causes the intricate specific shape of the active site. The substrates are held in the active site by forming temporary bonds with the hydrophilic R groups of the enzyme’s amino acids.

Here we have a diagram showing the ‘lock and key’ theory of enzyme action.

The substrate (key) is shown fitting perfectly into the active site (lock). The substrate binds to the enzyme and forms an enzyme-substrate complex. The reaction then takes place immediately and a substrate is either broken down into two or more products (catabolic reaction) or two or more substrates are bonded to make one product (anabolic reaction). The ‘induced fit’ theory states that initially the active site is not the perfect shape of the substrate but as it approaches, the shape of the active site changes to make the perfect fit.

Below is a sketched graph showing how enzymes reduce the amount of activation energy required for a reaction to occur; and hence speeding up the reaction.

This explains why enzymes are so vital in the human body. Some of our bodies reactions take place unanalysed at temperatures over 40*C which would damage our molecules unless we had enzymes to lower the heat energy required.

Preliminary work

Using the method explained in the “planning” section, preliminary tests were done to ensure that what needs to be done in the actual experiments is conducted both correctly and precisely. Initially, the ranges of the concentrations were 20, 40, 60, 80 and 100M and the amount used for each was 15ml. The amount of the processed liver used was 1g and each experiment was timed for 60 seconds (1 minute).

Conclusions drawn from preliminary experiments

We discovered that…

Timing the experiment for 60 seconds was far too long in the sense that some reactions finished faster than expected, especially the higher concentrations, i.e. 80 and 100M where reactions occur at faster rates.
In some experiments, particularly of those of 80 and 100M concentrations, the reactions finished far too soon, and released oxygen at a very fast rate into the measuring cylinder, thus making the recording of how much oxygen was collected very difficult as all the water had been displaced. To solve this problem, a larger measuring cylinder ...