The effect of pH on the activity of catalase

Introduction

Enzymes are biological catalysts. Catalase, an enzyme found in many tissues, catalyses the breakdown of hydrogen peroxide (H2O2) into water and oxygen. It is one of the fattest known enzymes.

2H2O2 catalase O2 + 2H20

Enzymes speed up reaction but not involved in the reaction. They provide a surface for reaction to occur. They also work very rapidly in reactions.

Observation

Enzymes are globular proteins that increase the rate of specific biochemical reactions; they act as organic and biological catalyst. Enzymes are specific for the reaction they catalyst. Each enzyme operates on a limited number of substrates of similar to generate product under well-defined conditions of concentration, pH, temperature etc.

Hypotheses

I predict that catalase will work at its optimum at pH 7 to 8. The optimum pH for most enzymes is neutral that is pH 7.Catalase will be denatured at in acidic conditions as the globular three dimension shape will be tortured and permanently be destroyed.

Requirements

This experiment will be successful done with these materials: Buffer solutions at pH 4.4, pH 5.2, 6.5 and pH 7.5.you will also need a pod, a burette, a pipette, Hydrogen peroxide, syringe, stop clock, test tubes, stoppers,

Safety measures

Hydrogen peroxide is an irritant product; it bleaches the skin instantly as it gets to it, therefore extra care must be taken when using it. Also care must be taken not pour water in pod or food the table you are working on.

Procedure

Nearly 50cm³ of tab water was filled into a burette and the burette was clamped upturned to a burette stand while its head was dipped into a pod containing water.

5cm³ of buffer solution (ph 4.4) was transferred into a test tube and then 3cm³ of yeast was added to it. A bung conserved the test tube over it having a delivery tube on it.

The delivery tube was then placed under the burette, 1cm³ of hydrogen peroxide was then measured in to a syringe and the syringe was connected to the test ...

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Procedure

Nearly 50cm³ of tab water was filled into a burette and the burette was clamped upturned to a burette stand while its head was dipped into a pod containing water.

5cm³ of buffer solution (ph 4.4) was transferred into a test tube and then 3cm³ of yeast was added to it. A bung conserved the test tube over it having a delivery tube on it.

The delivery tube was then placed under the burette, 1cm³ of hydrogen peroxide was then measured in to a syringe and the syringe was connected to the test tube.

Hydrogen peroxide was injected into the test tube (Injecting hydrogen peroxide into the test tube before positioning it under the head of burette infiltrated the air that should not go to the burette).

The stop clock was immediately started immediately and every 15 second the change in volume of the water in the burette were measured. This was done for 3minutes and the clock was then stopped.

The procedure was then repeated for buffer solutions pH 5.2, 6.5 and pH 7.5.

Results

My group results

Discussion and analysis

The experiment was done using different types of buffer solutions. A buffer solution is one which resists change in the H+ concentration (pH) on the addition of an acid or alkaline. Changes in pH alters the ionisation state of amino acids, so if ionic bonds are important to structural stability then the shape of the enzyme will change and the functionality of the enzyme will also change. This is a general phenomena related to the overall three dimension shape (3-D) of all enzymes.

From the class mean results I can see that pH 4.4 produced a volume of 13.87, between 15 sec and 60 sec, the line was the most steepest which indicates that the rate of the volume produce in the greatest, after 60 sec the rate begun to slow down as the line was less steep. The activation energy for catalase at this point was a bit low so made the reaction much slower.

At pH 5.2 the reaction begun to increase, more gas was produced which was 14.60 at the end of the 3mins., the reation was much quicker than that of pH 4.2, this was because there were more kinetic energy for the catalase to move rapidly and therefore colliod more with the substrate to form an enzyme-substrate complex.

At pH 6.5, the volume produced at the end of the 3 mins, was 14.09, but from the class mean graph, there was a steep down, the graph shows that pH 6.5 volume was lower than that of 5.2. Tere are so many factors that could contribute to this anomally, one of the factors may be inaccurate measurement of the yeast and the hydrogen peroxide, it may be accurate in my group, but be inaccurate in diffierent group. It could be that a person have put more amount fo yeast or hydrogen at different condition at pH5.2 than pH 6.5, example, someone could have measured 5.1ml fo yeast and use 4.9ml for pH 6.5. this then means that there will be more particles in the solution which makes high chance of collision to form enzyme-substrate complex and form more products.

At pH 7.5, the volume produced was extremely higher, it was 17.27, from the results catalase reached its optimum. From mine previous knowledge I know most enzymes work at their optimum pH which neutral i.e pH 7. It may also be that the enzyme catalase haven’t yet reached its optimum or had passed it optimum slightly, this would have be confirm or dealt with if say pH 7 and pH 8 was used in this experiment.

With reference to my error bars on mine graph, it is shown clearly how accurate the class mean was on each pH. It is also shown clearly that if I compare pH’s 4.4 and 6.5 to pH’s 5.2 and 7.5, I can tell that because the error bars on pH 4.4 and 6.5 are spreadout , the results are less accurate then that of pH’s 5.2 and 7.5 which are much much closer.

Evaluation

Overall I think my hypothesis was correct and the experiment proved it. There are so many factors that could contribute to some improvement in the method. One isa gas syringe could have been used to measure the volume from the burette was turned upside down and many people found it difficult reading the the volume taken at each time interval. Time could also cause a be error as time may be different at diferrent times and redings may be inaccurate.

Another cause of an error in this pratical could be reading the volume on the burette, this was because the readings were white and was almost the same colour as the water and therefore made it difficult to read and record accurate volumes. This could be eliminated next time by using burettes with coloured readings when repeating this practical.