Factors Affecting the Action of Copper Sulphate On theActivity of Enzyme (Catalase) Reaction.

FACTORS AFFECTING THE ACTION OF COPPER SULPHATE ON THE ACTIVITY OF ENZYME (CATALASE) REACTION.

AIM

The aim of the experiment is to observe the factors that affect the rate of a chemical, the activity of the enzymes and the volume of oxygen produced when an inhibitor is introduced into the reaction.

We know that when hydrogen peroxide (substrate) is being catalysed by an enzyme (catalase), it gives product of both oxygen and water. Based on this knowledge we have to determine the effects of temperature, concentration and the PH, as well as how they affect the volume of oxygen produced.

2H2O2 + CATALASE = H2O + O2

PREDICTION (HYPOTHESIS)

The substance, which is needed to react with the mixture of hydrogen peroxide and catalase, is copper sulphate (CU2SO)

I predict copper sulphate will reduce the rate of reaction between hydrogen peroxide and catalase because it is an inhibitor based on my knowledge. Inhibitors slow down or stop the reaction by competing with the substrate (2H2O2) for the active site of an enzyme (catalase) in due cause slowing down rate of reaction.

On the other hand it may act as a cofactor because of the ions which they can produce. Cofactors are non-protein substances, which aid the catalytic action of an enzyme.

INTRODUCTION

In order to carry out experiment in the investigation, in a successive way, good background knowledge of the rate of chemical reaction occurrence is needed with the factors, that affects it and the order in which they make an effect.

Enzymes are globular proteins which are classified as biological catalyst, which speed up/control the rate of a chemical reaction or metabolic reaction in our body without altering the substrates nor products neither does it alter the equilibrium between them without itself undergoing any changes.

Reactions will still occur without enzyme, only they would occur too slowly or not at all. All human activity and chemical reactions would take months if not years while enzymes, which are made by all living organisms, makes it a lot faster by decreasing the activation energy/overcoming enthalpy.

Enzymes are made up of tertiary proteins, which consist of long chains of amino acids. Amino acids are held together by hydrogen bonds, ionic bonds, and disulphide bridges in its three dimensional shape. The three-dimensional allows enzymes to form receptor sites for it’s for its substrate. The shape of the enzyme changes depending on its immediate environments. Change in PH, temperature can change its three-dimensional shape.

Enzymes are specific in structure and function because they can only catalyse one particular substance, which fits into their active sites.

Active sites are the smallest functional part of an enzyme, which can be called a cleft, contour, or depression, which comes in contact with the substrate. The active site is made up of 3-12 amino acids held together by some bonds of hydrogen and others.

LOCK AND KEY THEORY

Lock and key theory is the theory which states that the active site are like lock while substrates are keys with the same shape or identical to the shape of lock that fits exactly into he lock to open it, meaning that active sites are specific in shape and function, and only substrate with the exact shape function can only it in, and they are held by temporary forces until it is catalysed and released.

INDUCED FIT THEORY

Induced fit theory states that not all substrates or activities of an enzyme match perfectly or fit exactly ...

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INDUCED FIT THEORY

Induced fit theory states that not all substrates or activities of an enzyme match perfectly or fit exactly in their shapes but are at times slightly different in shapes, but during the course of reaction, one of them, mostly enzyme change its shape by moulding itself around the substrate, at times the substrate does adjust to the shape of the enzyme active site. While they are held together by some temporary forces, the active site of an enzyme puts a stain on the substrate molecule and then lowers the activation energy.

Induced fit theory is the latest version or model of LOCK AND KEY THEORY. Those changes can only occur because all enzymes are flexible and are stable on their 3-D structure.

Activation energy is the energy needed to start or boost a chemical reaction. It is needed to break down the existing bond inside molecule.

When a chemical reaction is shaken slightly or heats being applied to it, the kinetic energy of the particles are increased and so they move faster than before, making collision chances greater and rate of reaction increases.

TEMPERATURE

When a chemical reaction is heated the rate of the chemical reaction is increased. Heating gives more kinetic energy to molecules and they move quickly. Therefore there is a greater chance of collision between molecules and the rate of reaction is increased.

The impact when they collide would cause a reaction because they are moving at a greater speed.

The increase in the temperature of an enzyme-controlled reaction would bring about an increase in the rate of reaction, up to a certain point or degree. Increasing the temperature, of some enzymes to 40OC, increase the rate of reaction, due to the increase in kinetic energy and substrate energy of the enzyme molecules.

When the rate of enzyme catalysed reaction reaches a peak at a particular position, it is called optimum temperature.

After optimum temperature any further increase in temperaturecauses the vibration of the atom of the enzymes creating the breakage of hydrogen bonds and others as well that holds the particular enzyme molecule in its tertiary structure, with its specific shape and it causes

A change in tertiary structure of enzymes

It alters its 3-D shape, including its active site, which no longer fits the substrate and when this does happen it is said that the enzyme has denature.

The change is permanent and can not be reversible by cooling and its active site has been lost since it can no longer form enzyme-substrate complexes.

Cooling below optimum temperature on the other hand in activates the enzyme but the enzyme can work faster again when it is warmed up.

PH is the number of hydrogen ions in a given solution.

Enzymes do have an optimum ph at which they do work very efficiently. Small changes in ph range of an enzyme can cause denature. Acidity and alkaline can change the active site of an enzyme.

Free hydrogen (H+) or hydroxyl ions (OH-) can affect the changes on the amino acid side chain of the enzyme active site. This will affect the hydrogen bonding and so change the three-dimensional shape of enzyme and the shape of active. The substrate no longer fits the active site, the enzyme losses its activity. If the active site is flooded with either hydrogen ions or hydroxyl ions, it can prevent the enzyme and substrate from fitting together

CONCNTRATION OF ENZYME

When there is suitable temperature, PH condition and an excess substrate, then the rate of reaction is directly proportional to enzyme concentration.

When the concentration of enzyme is low and more concentration is added to it, the initial rate of reaction will increase in, when more enzymes is added at that period it is more likely that a substrate will bind to the empty active site on the enzyme. At a higher enzyme concentration, adding more enzymes has no effect on the initial rate of reaction in that the active site of enzyme is fully occupied.

SUBSTRATE CONCENTRATION

If the amount of enzyme stays the same, the rate of reaction will increase with an increase in substrate concentration, up to a point.

When the enzyme active site are all working as quickly as they can, adding more substrate does not have an increase in the rate of reaction.

METHOD

In this experiment, observations are very much essential, so making observation has to be careful and fast as the reaction is very fast in the experiment.

To observe the effects of CUSO4 on the activity of catalase a controlled experiment is needed that is an experiment without CUSO4. It is necessary because it is with this experiment that comparison is made with the experiment that is about to be done.

When the experiment with CUSO4 is carried out without a controlled experiment, the effects of CUSO4 won’t be known, in that the effect of Catalase on hydrogen peroxide without CUSO4 is unknown and comparison can not be made.

The concentration of some of the substances about to be used has to be varied while others have to be fixed.

VARIED VARIABLES

The concentration of hydrogen peroxide will be varied
The concentration of water will vary for different concentration of H2O2

FIXED VARIABLES

The concentration of yeast will be fixed
The concentration of CUSO4 will be fixed as well.

Some concentration will be fixed while others will be varied because others are of relative importance to the other.

DEPENDENT VARIABLES

Oxygen (O2)

INDEPENDENT VARIABLES

Hydrogen peroxide
Water.
Yeast
CUSO4

To start the experiment, water of different volume, should be added to varying volume of hydrogen peroxide, which are in a test tube, to reduce the concentrations of hydrogen peroxide. The different concentrations will be placed in test tube rack. Different volumes of yeast will be poured into test tubes and placed in racks. The different concentration of hydrogen peroxide will each be poured into conical flasks with a delivery tube connected to it and then sealed with a bung. The other end of the delivery tube is passed into the mouth of the gas syringe; the syringe is of measurement 100cm3. A stop clock is at hand ready to start. Immediately the yeast is injected into the solution the time should start and the reading of the amount of oxygen should be taken because the reaction starts immediately the yeast is injected into solution.

The experiment should be carried out twice or more than twice for each concentration of hydrogen peroxide so that the errors and mistakes made during the experiment would be calculated out while taking the averages. I have chosen to carry out the experiment twice as time is against me.

During most of the experiment, shaking was applied as it took a while for it to start. This shaking raised the kinetic energy of the molecules of the reactants; making the collision between molecules of reactants more frequent and this increases the rate of reaction. The amount of oxygen produced is consistent. The PH we assumed to be the same for the experiments carried out

The same substances used for the controlled experiment should also be used for the investigation experiment. The amount of yeast (Catalase) used for this experiment will remain as the amount used before. The amount of CUSO4 to be used will remain fixed through out this experiment, the only varying substances will be hydrogen peroxide and water used for dilution. The procedure as used above will be followed except for the introduction of copper sulphate (CUSO4).

The different volumes of different concentration of the solution of hydrogen and water will be poured into a conical flask. The appropriate amount of copper sulphate would then be added unto it, and then sealed with a bung with delivery tube in place and passed into to the mouth of the gas syringe. Yeast is then added into the solution and timing starts immediately. The amount of gas (oxygen O2) evolved is noticed on the measuring cylinder as it displaces water and recorded.

Before the experiment is ever started the dilution table should have been drawn, containing all the volumes of the reactant mixtures for both the controlled and with copper sulphate. After the dilution table has been drawn the result table should also be drawn to record all the volumes of oxygen given off every ten seconds (10s).

I have chosen to measure the amount of oxygen produced every ten seconds because it is a very fast reaction and the amount of oxygen produced would decrease as the time gets prolonged because collision rate would have decrease as well.

APPARATUS

Conical flask

Delivery tube

Test tubes

Stirrer

100cm3 gas syringe

10 measuring cylinder

Big measuring cylinder

Clamp

Bath

Bung &

Stop clock.

The diagram is shown below.

This experiment would be a fair experiment because it can be repeated. If the experiment is carried out its wrong the concentration can easily be changed. The experiment would also be fair because the yeast and copper sulphate are fixed.

The timing in this experiment should be, it is also very necessary because if the timing should go wrong the trend in the experiment won’t Colette.

I felt this apparatus was easy to set up without much difficulty. The apparatus was also easy to take the measurements during my trial experiment while in the other apparatus it was hard taking reading from it. It would also be difficult to quickly start an experiment once a mistake is made because it is hard to set up.

SAFETY AND ACCURACY

During this experiment care should be taken when handling fragile equipment’s, which includes all the apparatus. When they break they can cut the badly causing serious injury.

When handling hydrogen peroxide be careful as it can burn the skin when in contact.

Hydrogen peroxide can be irritating to the skin and eyes. To avoid irritation, put on lab coat and goggles and also wash away with water immediately in contact with your skin.

Hydrogen peroxide can also be bleach, so put on lab coat always to avoid your clothes getting bleached.

ACCURACY

Measure the correct volume of the substances, as the slightest hinge in measurement could lead to error or anomalous result

Make sure the measuring cylinder is upright and not in an angle, because the measurement could be incorrect when tilted.

The volume should be recorded every 15s.

Don’t contaminate the substances as it could lead to wrong result and errors.

During my trial experiment the range of concentration was very close, making the effect hard to detect. During one of the experiment, the result was anomalous because while we were trying to start the experiment, we used warm water in rinsing the conical flask. This then gave the rise in temperature and increased the rate of collision in the reaction making the result higher.

This particular experiment was with copper sulphate, so the result was higher than that we had without copper sulphate.

In my next experiment I wish not to repeat that same mistake but to correct it and make my result accurate and valid.

CONCLUSION

The substance which effect was needed was CUSO4. I can now say it is an inhibitor because during the trial experiment it slowed down the rate of chemical reaction. When the concentration of hydrogen peroxide is higher than that of copper sulphate the rate of reaction is not that badly affected.

Temperature I believe has the most powerful effect if we are looking at the factors that affect enzyme activity. It is then followed by the concentration and then it is followed by the pH.

Apparatus