Experiment To Investigate The Effect Of Concentration On An Enzyme Based Reaction.

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Autumn 2003

Experiment To Investigate The Effect Of Concentration On An Enzyme Based Reaction

Aim:

        To investigate the effect of either enzyme or substrate concentration on the rate of decomposition of hydrogen peroxide using catalase enzyme.

Introduction: (information obtained from “OCR Biology 1”, “Advanced Biology” & “Hutchinson Dictionary of Science”)

An enzyme is a protein molecule that speeds up chemical reactions in all living things. Without enzymes, these reactions would occur too slowly or not at all, and no life would be possible. Enzymes, for example, are needed for respiration to occur. 

Enzyme molecules function by altering other molecules, known as the substrate, to form a new product. They combine with the substrate molecules to form a complex molecular structure in which chemical reactions take place before the enzyme, which remains unchanged, separates from the product of the reaction. There have now been over seven hundred different enzymes identified by scientists, which have varying turnover numbers between 100 and several million. This ‘turnover number’ refers to the number of substrate molecules that one of the enzyme molecules can turn into a new product in one minute.

Enzymes are classified into several broad categories, such as hydrolytic, oxidising and reducing, depending on the type of reaction they control. Hydrolytic enzymes accelerate reactions in which a substance is broken down into simpler compounds through a reaction with water molecules, oxidising enzymes, known as oxidisers, accelerate oxidation reactions and reducing enzymes speed up reduction reactions, in which oxygen is removed.

The enzyme used in this experiment, catalase, occurs naturally in the tissues of most living organisms and can be found in the liver of the human body. It is present in the peroxisomes, which are micro body organelles that house various oxidation reactions in which toxic peroxides are generated as side products, of nearly all aerobic cells. It serves to protect the cell from the toxic effects of the side product, in this case hydrogen peroxide (2H2O2), by catalysing its decomposition into oxygen and water and is the fastest known enzyme with a turnover number of 6 million.

        Catalase is also found in yeast, to be used in this experiment, as well as other natural sources including potatoes and celery, whilst hydrogen peroxide does not only occur naturally. It can be made chemically and is used as bleach, a mouthwash and, in medicine, as an antiseptic to remove dead tissue from wounds and ulcers.

        The equation of the reaction between catalase enzyme and hydrogen peroxide substrate is as follows:

Hydrogen peroxide + catalase  Oxygen + Water

2H2O2                                                         O2       + 2H2O

        This breakdown will occur most efficiently in conditions most similar to those in the human body, i.e. at a temperature of approximately 37˚c, and at a pH created by the two solutions, which will be approximately neutral.

Identification of Variables:

Possible Variables:

        These are the conditions that could be varied when investigating the effects certain environments have on the rate of breakdown of hydrogen peroxide by catalase found in a yeast solution:

  • Concentration of yeast solution (enzyme) – When there is an abundance of substrate, the more enzyme there is available, the faster the rate of the reaction will be as it would be more likely for an enzyme molecule to collide with a substrate molecule at the correct angle and with sufficient force for a reaction to occur.

However, were there to be a limited amount of substrate available, even if more enzyme molecules were added (the concentration increased), there would not be enough substrate molecules to go round. This would cause a levelling off of the rate as no more reactions would be possible as all the substrate molecules would be locked into an active site, although in practice this would be difficult to achieve as you would need a very high concentration of enzyme compared to substrate to achieve this.

  • Concentration of hydrogen peroxide (substrate) – The more substrate molecules there are available, the more often a reaction inducing collision with an enzyme molecule will occur, and the faster the rate will be.

However, if there are only a limited number of enzyme molecules available, this pattern will not continue. Once all the enzyme molecule active sites are filled, adding more substrate will not increase the rate, the molecules will only be left ‘queuing up’ for empty active sites, causing the rate to level off.  

  • Pressure on experiment solution – Increasing the pressure under which the reactions between the enzyme and substrate molecules occur will increase the rate, but, as with the previous two variables, only up to a point. As the pressure increases, the molecules of both solutions will be forced closer together, which will increase the chance of a reaction inducing collision with the substrate molecule fitting into the active site. There is a limit as to how close together the molecules will fit and, once that limit is reached, the rate of the reaction will level off as it will not be possible for the molecules to collide with each other any faster.
  • Temperature of experiment solution – raising the temperature will increase the rate of the reaction as it gives the molecules more energy causing them to move faster, making them more likely to collide successfully to induce a reaction. Further more, as they have more energy they are more likely to be able to overcome the activation energy barrier when they do collide. In most enzyme based reactions, a temperature increase of 10˚c doubles the rate of the reaction, up to a point. As enzymes are proteins they become denatured at high temperatures when the hydrogen bonds and hydrophobic interactions holding the active site in shape break. Once this occurs, substrate molecules no longer fit into the active sites and reactions cannot take place. As the temperature continues to rise, all the enzyme molecules become denatured and the reaction stops.

The peak, or optimum, temperature for enzymes that act in the human body is 37-40˚c, for a plant is about 25˚c and for bacteria / a fungus is up to 80˚c. Enzymes in the human body, such as catalase, which is to be used in this investigation, denature by approximately 60˚c.  

  • pH of experiment solution – A change in pH affects the structure of the enzyme molecule, making it harder for a substrate molecule to bind with it as the active site will change shape. pH affects the ionic bonds in the enzyme structure as well as the ‘R’ groups that line the active site with which the substrate physically binds.

Enzymes normally have a very narrow pH range over which they are most effective, and the optimum pH tends to be neutral (7), but enzymes such as pepsin, found in the acidic conditions of the stomach, work best at pH2, a point by which most other enzymes would become denatured.

Control Variables:

        These conditions will remain constant throughout the investigation, which will ensure a fair test and increase the accuracy of the results:

  • Concentration of hydrogen peroxide (substrate) – The initial concentration of 20vol. will not be diluted with water and all the substrate will come from the same source so that if there is any small inaccuracy in the composition of the concentration, its effect will be consistent.
  • Pressure on experiment solution – The experiments will all be conducted in the same room under the same conditions, which will maintain a constant pressure.
  • Temperature of experiment solution – Similarly, the experiments will take place at 20˚c, close to room temperature and therefore easier to maintain accurately than a warmer temperature, to remove inaccuracies. Further more, as catalase acts so fast, completing the experiments at room temperature will decrease the rate (room temp. is below optimum body temp.), making the timing of the reaction easier.
  • pH of experiment solution – As the same components will be added to the experiment solution each time, the pH will remain the same throughout the experiments.
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Independent Variable:

        In this investigation the independent variable, the condition to be changed, is the concentration of the yeast solution. The range to be used is 0, 2, 4, 6, 8 and 10%. These concentrations were calculated using the table below:

Dependant Variable:

In my investigation I will measure the volume of oxygen given off in 20 seconds for each concentration of yeast solution. The start point for the taking of this reading will be identified by when the hydrogen peroxide is added to the yeast solution in the boiling tube.

Biology Coursework

Sharon Dulai ...

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