Investigation of how different concentration of enzyme catalase affects the rate of breaking down substrate hydrogen peroxide

Introduction

Enzymes are large proteins that speed up chemical reaction. As globular protein, enzymes have a specific three-dimensional shape which is determined by their sequence of amino acids. Despite their large overall size, enzyme molecules only have a small region that is functional. This is known as enzyme’s active site. The substrate molecule is held within the active site by bonds that temporarily form between the R groups of the amino acids of the active site by bonds and all groups on the substrate molecules. This structure is known as enzyme-substrate complex.

Enzymes are classified into several categories, such as hydrolytic, oxidising, and educing. Depending on the types of reaction they control. In this case, the enzyme I will use in the investigation is catalase from celery extract, which is concluding as hydrolytic enzyme. This type of enzyme accelerates reactions in which a substrate is broken down into simpler compounds through reaction with adding up water molecules. Oxidising enzyme, known as oxidises, accelerate oxidation reaction; reducing enzyme speed up reducing reactions in which oxygen is removed.

A substrate is the molecule, which can bind into the active of enzyme. In this case, I will use hydrogen peroxide as the substrate.

Enzyme works in the same way as a key operates a lock. Enzymes’ active sites have a particular shape like a lock and only a particular key ( substrate) can fit into that lock. Enzymes are therefore specific in the reactions that they catalyse. This is known as the ‘lock and key theory’. In practice, unlike a rigid lock, the enzyme actually changes its form slightly to fit the shape of the substrate. In other works, it is flexible and moulds itself around the substrate. As it alters its shape, the enzyme puts a strain on the substrate molecule and thereby lowers its activation energy.

In this case, enzyme catalse has a specific active site, which just for the substrate hydrogen peroxide to fit in, then the reaction takes place. The reaction involved is hydrolysis.

The equation of reaction is:

2H2O2 2H2O + O2

Factor that would affect the reaction:

Temperature

At low temperature, the reaction takes place very slowly, this because molecules are moving relatively slowly as have low kinetic energy. Substrate molecules will not often collide with the active site, and so binding between substrate and enzyme is a rare event. Therefore, reaction is slow. A rise in temperature increases the kinetic energy of molecules which therefore move around more rapidly and collide with one another more often. This means at a higher temperature, the reaction will take place faster than a lower temperature and an increasing or a decreasing of temperature will affect my reaction and results. For this reason, I will control this effect by using a water bath to maintain the temperature the same during the whole reaction. As enzyme work best at a certain temperature, this is known as optimum temperature. If the temperature is too low, enzyme cannot work properly; if the temperature is too high, this may denature the enzyme active site and enzyme will lose its function. So to avoid this problem, I will keep the water bath has the temperature of 25°C, which is the room temperature. Therefore enzyme catalase will work properly and temperature will not be a factor to affect this investigation.

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Most enzymes also have an optimum pH at which they function best. In human body, most enzyme work fastest at an optimum pH of about 7. For example, the digestive system Pepsin found in the stomach to digest proteins. A change I pH means a change in the concentration of hydrogen ions in the surrounding of the enzyme. This affects the ionisation of R group in the amino acid residues of the protein molecule and the shape of the active site to bind with the substrate. The lower the pH, the bigger the hydrogen ions’ concentration it is . Hydrogen ions can interact with the R group of amino acids, affecting the way in which they bond with each other and therefore affect their 3D shape. So the lower the pH, the higher the hydrogen ions’ concentration it is, and therefore the slower the reaction. Oppositely, the higher the pH, the faster the reaction it will take place. To control this factor, I will keep the pH all the way through the same by using a pH paper to check the pH number during the experiment.

Enzyme concentration

The rate of reaction increases as the concentration of enzyme increase. Because higher concentration of enzyme means higher number of enzyme molecules, so that more enzyme molecule will collide with substrate molecule, therefore the reaction will take place faster. When there is plenty of substrate, the rate of reaction is not limited by the concentration of enzyme. Therefore if the concentration of enzyme is increased, the number of collision with the substrate molecule and hence the rate of reaction will increase. If the amount of substrate is limited, the rate of reaction decreases because if the amount of substrate is limited, the enzyme’s active site will not collide with substrate very often, therefore a few products will be made. As the reaction progressing, the substance will be broken down slowly, therefore less substrate left. So enzyme molecules freer, therefore products will be produced more slowly. Hence the slower the reaction goes. In this experiment, the enzyme concentration is an independent variable which I will vary the concentration of celery extract each time to investigate how different concentration of celery extract( enzyme concentration) affect the rate of breaking down hydrogen peroxide.

Substrate concentration

Increasing in the substrate concentration will increase in the rate of reaction. Because increasing the substrate concentration, we increased the number of substrate molecules; therefore more collision will take place between substrate and enzyme’s active site. Hence mere products will be produced. Therefore the faster the rate of reaction takes place. However, if we continue increase the substrate keeping the enzyme concentration and volume the same. The rate of reaction will not continue to increase. This is because a higher substrate concentration active site of all the enzyme molecule is busy with substrates. The number of enzyme molecules because a limiting factor. The rate of reaction at higher substrate becomes constant. In this case, I will keep the volume of hydrogen peroxide the same for using each time.

Inhibitors

Enzyme inhibitors are substances that directly or indirectly interfere with the functioning of the active of an enzyme and so reduce its activity.

There are two types of inhibitors:

Competitive inhibitors

Competitive inhibitors are the molecules which have a shape similar to the substrate, so they fit into the active site of the enzyme. Therefore the substrate cannot enter into the active site and so the enzyme cannot catalyse the reaction. Hence, the reaction is slow down. However, the inhibitors are not permanently bound to the active site and so, when it leaves another molecule can take its place. Sooner or later, all the substrate molecules will find an active site, but the greater the concentration of inhibitors, the longer the reaction will be.

Non-competitive inhibitors

Non-competitive inhibitors will bind to another part of the enzyme rather than the true active site. This changes the shape of the enzyme’s active site so that the substrate no longer fits. Therefore the reaction is slow down. As the substrate and the inhibitor are not competry for the same site, an increase in substrate concentration does not decrease the effect of inhibitors.

To control the factor of inhibitors. I will not add any substance except celery solution and hydrogen peroxide during the experiment.

Prediction

I predict that the higher the concentration of celery extract I use, the fast the rate of breaking down hydrogen peroxide I will get.

As in higher concentration of celery extract, the more number of catalase molecules in the solution. So there are more collisions between substrate ( hydrogen peroxide) and enzyme( catalase) active site, and more products will be formed( water and oxygen). The rate of reaction can be measured by measuring the volume of oxygen produced in a period of time. The larger the volume of oxygen produced in the same period of time, the fast the reaction is taken place.

Preliminary work

In order to get finest and reliable results, I did a preliminary work. This enable me not just find out a suitable range of celery extract of catalase concentration I will use in my real experiment, but also help me descried the time interval I will measure the oxygen get and justify the equipment and method. I think these all lead me to get reliable and accurate results in the end.

In the preliminary work, I choose to do three different concentration of celery extract. To get different concentrations of celery extract, I used distilled water to dilute the celery extract.

I tested the range of celery extract between 100% --20%, the highest concentration is 100%, the lowest concentration is 20% and the median is 60%. The reason I chose these three concentrations is because between each concentration, there is 40% difference and this enable me to test that in which level of concentration, reaction goes best and to find out which concentration of celery extract I will use in my real experiment.

The concentration of celery extract I used as following table:

I used 15 cm3 of hydrogen peroxide for each concentration of celery extract, therefore the concentration of substrate will not affect the rate of reaction.

The results of preliminary work as following:

From my preliminary result, I found that at 20% celery extract, there is not much gas produced, the volume of oxygen produced is 4.4 cm3 and the measure equipment gas tube cannot measure the volume accurately if the volume of gas less than 1 cm3 . At 100% celery extract, I found that the reaction takes place fairly fast and finished at 100s which was before 3 mins. The volume of oxygen at 100s is 44.8 cm3 . Comparing to other two concentration of celery extract breaking down hydrogen peroxide to produce oxygen volume at 100s, the volume is much bigger and I can tell the reaction at 100% celery extract went fastest as the volume of oxygen produced the most at the same period of time as 20% and 60%. At 60% celery extract, the reaction went very well, the amount of oxygen steadily increase and I can tell the reaction take place faster than 20% by comparing the volume of oxygen. The volume of oxygen produced in the final is 14.6 cm3 which is much bigger than the volume of oxygen produced at 20% , but compare the volume of oxygen at 100% at 100s, the volume at which 100s of 60% celery extract is 11.6 cm3 which is much smaller than 100% celery extract. So I can say the reaction at 60% is slower than the concentration at 100% celery extract.

In the preliminary work, I decide to increase the volume every 10s, this enable me to regulate the results and get many points to plot a nice graph. Also because for 100% celery extract, the reaction take place very quick, measure the volume every 10s can allow me to get at least 5 points to plot a graph.

In my real experiment, I decide to use the range of concentration between 25%---100% celery extract, the concentrations I will use are: 25%, 40%, 55%, 70%, 85%, 100%. I conclude from my preliminary work that there was no need to carry out the experiment for less than 25% because it would happen too slowly to be bothered with. I decided to choose concentrations that had less of a difference so that when it comes to plotting my graph I will be able to plot points closer together and get a more accurate line of best fit. Therefore more accurate rate of reaction I will get.

I will use 20 cm3 of hydrogen peroxide in my real work instead of 15 cm3 used in preliminary work. Because we want make sure the factor of concentration fo substrate will not affect the rate of reaction, therefore the substrate should be excess. So 20 cm3 hydrogen peroxide will be used in my real work to ensure that I have excess substrate therefore it will not limit the reaction.

In my real work, I will repeat the experiment for 3 times therefore I will be able to find the average volume of oxygen for each concentration of celery extract by adding the three times for one time interval and then dividing by three. Finding the average will eliminate any anomalous results, making the data more reliable. By using these average data, I will be able to plot a graph of which oxygen gas produced against the time taken. A line of best fit will be drawn through each point, and I can find the rate of reaction by calculating the initial rate of reaction over first 30s. the initial rate of reaction will be calculated by the volume of oxygen at 30s divide by 30s.

Also, it is very important to take into considering on the safety precautious. This involves wearing goggles for eye protection, especially when we use hydrogen peroxide because it is toxic. As most apparatus are made of glasses, so it is necessary that do not touch any glasses pieces if they broke. Wearing a lab coat is also an important issue. This protects our skin or clothes from getting in contact with the chemicals being used.

Apparatus list and justifications

celery extract: this contain enzyme catalyse and will be diluted with distilled water to make the different concentrations of catalyse solution. All celery extract should be made from one patch and this enable the all the 100% celery extract have the same concentration.
Hydrogen peroxide: this is substrate in the experiment and is broken down into water and oxygen by enzyme catalase. And the product oxygen can be used to measure the rate of reaction.
Distilled water: this is better than using tap water because this is pure and does not contain any other ions which would affect the reaction.
Syringes( for the following types, on syringe for hydrogen peroxide and one for celery extract)

2ml syringe: measure the volume of 1 cm3 -- 2 cm3

5ml syringe: measure the volume of 2 cm3 --5 cm3

10ml syringe: measure the volume of 6 cm3 -- 10 cm3

20ml syringe: measure the volume of 11 cm3 -- 20 cm3

Three glass beakers: one for celery; one for distilled water and on for hydrogen peroxide.
Conical flask: the special shape of conical flask allow hydrogen peroxide stir with the catalyse solution in one way or another to ensure that the enzyme and substrate molecules are evenly dispersed without any being lost in the process.
Thistle funnel with S-shape delivery tube: allow me to delivery oxygen to the tube without by moving the bump.
Clamp: this can make sure the meniscus keep horizontally.
Gas tube: gas tube will be used instead of a burette because gas tube has the scale in the positive way, so it is easy for me to read the volume of oxygen produced. But is the volume of oxygen less than 1 cm3 , this will not be able to read the volume.
Water bath: this can be used to maintain the temperature of the reaction, so that temperature during the reaction will stay the same and will not affect the reaction.
Stopwatch: to measure the time for oxygen produced.

Method

using the syringe to make up the following concentration of catalase using the distilled water and celery extract.

2. set up equipments as diagram. ( making sure there are no bubbles inside the gas tube)

using 20ml syringe to measure 20 cm3 of hydrogen peroxide and transferring it to the conical flask which has contain 25% of celery extract, make sure that the bottom of thistle funnel is below the liquid level.
once inject the hydrogen peroxide into the thistle funnel, put the delivery tube under the gas tube after few seconds and start the stopwatch immediately.
read and write down the volume of oxygen gas every 10s for up to 3 mins.
repeat step 3 to 5 for catalase concentration of 40%, 55%, 70%, 85% and 100%.
redo this experiment 3 times for each concentration of catalase.
calculate the average volume of oxygen and plot a graph.

Method justifications

I chose to use different concentrations of celery extract therefore I can get different concentration of enzyme catalase concentration. This enable me to investigate how different concentration of catalyse affect the rate of breeding down hydrogen peroxide. I will use 6 concentrations in order to plot a graph minimum of 5 points.

At step 4, the delivery tube is put under the gas tube after few seconds when hydrogen peroxide inject into the conical flask. This because the first few bubbles may come from the air in the delivery tube and are pressed out by the pressure. After few seconds to put the tube can avoid the fist few bubbles from the air.

The repeating lead me to get 3 volume of oxygen in each 10s, this enable me to see if is the result reliable. If the results are very similar, I can conclude that the are reliable; if it is not, perhaps the method is not good.( e.g. the delivery tube to gas tube may lose some bubbles when starting the stopwatch)