In this experiment, I am traveling to find the consequence of different concentration of enzyme catalase on the rate of reaction of decomposition of H peroxide. Normally, H peroxide is produced of course in human or works cell. Hydrogen peroxide is the byproduct of respiration. As an oxidant, it will break up to organize O and H2O. The chemical equation for the decomposition of H peroxide is 2 H2O2 a†’ 2 H2O + O2. The reaction is speeded up by the presence of enzyme, viz. catalase which is used in this experiment. This mechanism is of import in populating beings ‘ cells and organic structure system peculiarly in homo. This is because the caustic feature of H peroxide may damage the wall of liver where it is mostly produced during cellular respiration procedure. When it is present in high concentration, it is an aggressive and powerful oxidant, whereby it is unstable and besides risky as it will eat many substances including human tegument. Therefore, concentration of H peroxide in the cell should be invariably regulated. When H peroxide is used for the intent of experiment, this extremely caustic stuff should be kept in a container made up of non-reactive stuff such as glass. However, at low concentration, H peroxide can be used as germicide and antiseptic for medicative utilizations.[ 1 ]
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Diagram 1: Chemical construction of H proxide, H2O2
In this context, catalase, a teramer of four polypeptide ironss is made up of over 500 aminic acids long. It is besides categorised as ball-shaped protein in which the polypeptide concatenation is extremely folded into a compact spherical form. There is besides active site available to adhere to the H peroxide substrate to organize enzyme-substrate composite.[ 2 ]It is further adapted with four porphyrin haem groups to respond with H peroxide. Besides, the enzyme catalase is known to be one of the enzymes that possess a high turnover figure. Its turnover figure can be up to 600 000 whereby one molecule of enzyme catalase can catalyze the decomposition of 600 000 molecules of H peroxide to oxygen and H2O at organic structure temperature.[ 3 ]This reaction is known as katabolic reaction as the H peroxide molecule is broken down into O and H2O which are relatively smaller. Sometimes, catalase besides uses H peroxide to oxidize toxins including Phenols, Formic Acid, Formaldehyde and Alcohols. In this experiment, murphy is chosen to be tested due to the presence of catalase in it. However, other beings such as Fungis or barm can be used every bit good as they are manufacturers of enzyme catalase.
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Diagram 2: The construction of catalase
Enzyme is used to rush up the rate of reaction by take downing the activation energy of a reaction. Activation energy or free energy of activation, is the initial investing of energy for get downing a reaction – the energy required to deform the reactant molecules so the bond can interrupt for a reaction to happen.[ 4 ]Enzyme maps as biological accelerator in many chemical reactions that occur inside our organic structure. For illustration, saliva secretes enzyme amylase which catalyses the hydrolysis of saccharides in the oral cavity.[ 5 ]Not merely does enzyme play an of import function in keeping efficient map of organic structure system, it is mostly used in industrial field every bit good to rush up the production rate. For illustration, peptidase is normally used in biological detergent for domestic lavation and chymosin is used in industry of cheese. For an enzyme to transport out its map efficaciously, active site should show on the surface of the polypeptide concatenation. An active site is a channel or pocket formed by the folding form of the protein. This active site has peculiar chemical composing and electrical charges on the amino acids, which make up the specificity of the enzyme, in which it allows merely certain substances to adhere to it. When the substrates bind to the active site, here the working mechanism of enzyme starts. The binding of the substrate to the active site conveying the substrates closer and therefore AIDSs in bond formation in anabolic reaction. In katabolic reaction, the active site may falsify the form of substrate to interrupt its bond. When the merchandises are formed, the substances no longer suit into the specific form of the enzyme and will go forth the active site of the enzyme. The enzyme is free to adhere to another substrate and catalyse another reaction. The enzyme is non altered at the terminal of reaction.
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Diagram 3: The working mechanism of enzyme
As enzyme contains specific form and charge on its active site, its activity is easy affected by the alterations in the encompassing conditions. By and large, different pH, temperature, concentration of substrate or concentration of enzyme has a big impact on its efficiency in transporting out its map. Whenever the alterations in environing such as alteration in pH or temperature alter the bonding between the R group of the amino acids in the polypeptide concatenation which form the active site, the form of active site will alter and therefore the substrate will no longer adhere to the site. At this point, the enzyme is said to be denatured. On the other side, when the temperature or pH is optimal for the reaction, the rate of reaction is the highest. Although the optimal pH and temperature may change from one another, optimal temperature for most enzymes working in human organic structure system is frequently 37 °C. However, the presence of inhibitors or cofactors may change the enzyme activity every bit good. In this experiment, the consequence of enzyme concentration is chosen to be investigated on the rate of reaction catalysed by enzyme catalase. An addition in enzyme concentration will increase the active site available and therefore increase the rate of reaction until it reaches maximal speed when all active sites of the enzyme molecules are engaged.
Make different concentrations of enzyme affect the rate of reaction?
To look into the consequence of different concentrations of catalase on the rate of reaction to catalyze the decomposition reaction of H peroxide
To find the presence of catalase on the rate of reaction of H peroxide.
To develop effectual experimental accomplishments throughout the experiment
To find the consequence of different concentrations of enzyme on the enzyme activity
The higher the concentration of enzyme, the higher the rate of reaction until a maximal speed is reached.
Use a H2O supplanting technique to find the volume of O gas evolved
Calculate the rate of reaction by utilizing the gradient of the graph
Newly mashed or blended murphy, 3.0 % H peroxide solution, buffer solution ( pH 6.5 ) , distilled H2O
Boiling tubings, graduated tubings, 500 milliliter beaker, weighing balance, spatula, bringing tubing, halt ticker, mensurating cylinder, dropper, gum elastic spile, weighing dish
How the variable is determined
Concentration of catalase
By utilizing different mass of blended murphy at 1g, 2g, 3g and 4g. Different multitudes of blended murphy indicate the difference in concentration of catalase in its content.
The volume of O gas released
By entering down the reading on the graduated tubings at 30 seconds interval.
Volume and Concentration of H peroxide
By utilizing buffer solutions at pH 6.8 throughout the experiments
By utilizing the same volume and concentration of H peroxide, which is 2.5cm3 of 3.0 % H peroxide throughout the experiment
1 g of the freshly prepared or blended murphy is transferred into a boiling tubing.
5 cm3 of buffer solution is added into the tubing and it is swirled to blend the substrate.
A calibrated tubing is filled with H2O to the lip.
It is placed carefully into a beaker of H2O. One terminal of the bringing tubing is placed into the calibrated tubing with the other terminal with gum elastic spile ready to repair with boiling tubing.
2.5 cm3 of H peroxide solution is measured and it is added into the boiling tubing incorporating the murphy and buffer solution.
The tubing is instantly closed with a gum elastic spile connected to the bringing tubing. A stop watch is started by one member of the braces in carry oning this experiment.
The volume of gas released is measured for every 30 seconds for 5 proceedingss or until the gas development Michigans.
The experiment is repeated utilizing 2g, 3g and 4g of newly blended murphy.
The consequences obtained are recorded in a tabular array.
Graphs for volume of gas released against clip is plotted for each concentration or sum of enzyme used.
The initial rate of reaction for each concentrations of enzyme used are worked out.
Volume of O gas collected at murphy with mass of
Table 1: The volume of O gas collected at 30 intervals with different mass of murphy.
Formula 1: The average value of the day of the month obtained
Formula 2: The initial rate of reaction
Analysis of Data – Bar Chart:
Graph of O gas evolved against clip in different mass of murphy obtained
1g of blended murphy
78-24 = 54
Initial rate of reaction = Gradient of the graph
= 0.0611cm3 / s
2 g of blended murphy
17 – 6
Initial rate of reaction = Gradient of the graph
= 0.2895 cm3 / s
3g of blended murphy
Initial rate of reaction = Gradient of the graph
= 0.6579 cm3
4g of blended murphy
Initial rate of reaction = Gradient of the graph
= 0.7000 cm3 / s
Mass of murphy /g
Rate of reaction / cm3s-1
Based on the above experiment, the consequence of different concentrations of enzyme on the rate of reaction is successfully determined. Five graphs are plotted based on the consequences obtained in the experiment to demo the informations in a clearer manner and provides a better mean for analyzing. The consequences show that the rate of reaction is increased by an addition in enzyme concentration. In this experiment, murphy is used as beginning of catalyse. The first four graphs demoing O gas evolved against clip are drawn based on several mass of blended murphy used. The initial rate of reaction is measured from each graph by obtaining the gradient of the graph. A predicted line is drawn on each graph. By and large, the longer the clip taken, the higher the volume of O gas evolved. In the beginning, all graphs show an rapid addition, the velocity is the slow down as some of the substrates are converted to merchandises. For the substrate at 1 and 2 g of bent murphy used, the maximal volume of O gas evolved has reached within 300 seconds and a tableland is obtained. This is because the reaction has completed for all substrates. Theoretically, the maximal volume of O gas released should takes a shorter clip as compared to 1g and 2 g of murphy as more active site are offered. However, In the 3 and 4 g of blended murphy which react, the maximal volume of O is unable to be obtained within 300 seconds. This is likely due to some mistakes conducted throughout the experiment, peculiarly due to the vigorous and rapid reaction and in the procedure of altering the calibrated tubing. The mistakes will be discussed subsequently. The initial rate is taken because the rate of reaction is rapid as the hit between the substrate and enzyme is the highest. The rate of reaction may non be dependable to be compared between informations if readings are taken in the center of the experiment because some reactions have reached the maximal rate. The initial rate of reaction for H peroxide with 1g, 2g, 3g and 4g of blended murphies are 0.0611, 0.2895, 0.6579 and 0.7000 cm3/ s severally.
The initial rate of reactions for all the experiments are so compiled into the 5th graph. This shows a clearer image on the consequence of concentration of substrate on the rate of reaction. Initially, there is an addition in the rate of reaction when the mass of blended murphy additions. This is because the addition in the concentration of enzyme offers more active site for the binding of substrate. Then, the incline of increasing line becomes less steep with farther addition in concentration of enzyme. This is because the active site has been occupied by the substrates or it is said to be saturated whereby the addition in substrate has no farther consequence on the rate of reaction. Theoretically, the graph should make a maximal speed where the tableland occurs in the graph. However, in this experiment, the tableland is non shown because most likely the concentration of enzyme is non high plenty to adhere to all the 3.0 % of H peroxide substrate.
However, throughout the experiment some mistakes might happen in which the existent values may non be obtained. First, there is a high inclination for the reading obtained from H2O supplanting method to be inaccurate particularly when the volume of O gas evolved are excessively much that the first calibrated tubing is to the full filled with O gas and when the bringing tubing has to be transferred to the following prior-prepared graduated tubing. The bringing tubing reassigning procedure may devour some clip peculiarly if a gum elastic bringing tubing is used alternatively of a glass bringing tubing. This will do some of the O gas to get away into the H2O during the procedure. Next, parallax mistake may happen every bit good when the reading is taken from the graduated tubing on the volume of O gas evolved. This is because O gas is a colourless gas, in which its degree is non so clearly seen on the standardization of the calibrated tubing. To understate the mistakes, the experiment is repeated twice and the average reading is obtained. To farther increase the truth of the consequences, a piece of white paper can be placed behind the calibrated tubing to do the reading easier. Following, the possible mistake is greater if the experiment is carried out separately. This is due to the human limited ability to enter the reading and at the same clip ticker over the clip. Inaccuracy may originate. In this instance, a brace work is preferred in this experiment as one of the members times and the other one record the readings obtained. Following, when the mashed murphy is poured into the boiling tubing from the weighing dish, some murphy may be left in the weighing dish. To understate this mistake, a few beads of distilled H2O can be used to rinse the weighing dish to guarantee there is no residue left.
Consequently, there are a few safeguards that ought to be taken to increase the truth of the consequences obtained. For each experiment, the murphy used must be newly mashed or blended. If the murphy is prepared in a container, the palpebra of the container should be kept closed after the coveted mass of blended murphy is scooped out for each and every experiment. The readying of blended murphy in a beaker which is exposed to the air should be prevented because oxidization will happen and this may impact the activity of enzyme catalase in it. Changes in environing such as temperature may besides bring on alterations in the enzyme. A blended murphy is used alternatively of phonograph record of murphy so that it will respond easier. Its viscousness should be reduced so that it is easier to utilize. Next, H peroxide has to be stored in an opaque container as it breaks down rapidly when exposed to visible radiation. The palpebra of the container that contains hydrogen peroxide solution should be kept closed after each coveted sample is taken out utilizing a dropper as the O in the environing air may oxidize its content and causes the consequences to be inaccurate. A buffer solution is used to guarantee the pH is kept changeless throughout the experiment. The buffer solution of citric acid Na phosphate solution which has a pH of 6.8 is used because this is the optimal pH for the enzyme catalase. Furthermore, a H2O bath is preferred as the environing temperature may alter throughout the experiment. In add-on, as the gum elastic spile of the bringing tubing should be of the same size as the boiling tubing to guarantee all the gap of the boiling tubing incorporating enzyme and substrate is fit tightly, it should be pushed and twisted with attention. It should besides be checked from clip to clip to guarantee there is no escape of merchandise in gaseous signifier to the surrounding. Besides, the other unfastened terminal of bringing tubing should be placed in H2O all the clip for the bubble of gas to organize and lift to its surface. The presence of air bubbles guarantee that the gum elastic spile is still in contact with the boiling tubing unless the substrate and enzyme has wholly reacted. To repair the calibrated tubing in topographic point, a rejoinder base and clinch can be used. Besides, the boiling tubing incorporating reactants and enzyme ought to be swirled throughout the experiment to guarantee the substrate and enzyme react. This may increase the rate of hit between the reactants and enzymes and therefore fix the clip taken for the reaction to finish.
Throughout the experiment, some safety steps should be abided by. As the substrate used in this experiment which is hydrogen peroxide is extremely caustic, rubber glove should be used to protect the tegument. After the H peroxide is used, it should be disposed away and non to be returned to stock bottles as any contaminations may ensue in decomposition and detonation may happen. The blended murphies have to be handled carefully every bit good as it will annoy some people ‘s tegument. A lab coat should be put on. The glass wares and the bringing tubing used should be handled carefully as they are delicate.
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The hypothesis is accepted. The presence of enzyme increases the rate of reaction of H peroxide. When the concentration of enzyme additions, the rate of reaction additions until a maximal speed is reached.
The species of murphy
Different species of murphy may incorporate assorted concentration sof catalase
The age of murphy
An older murphy may hold lower concentration of catalase
The freshness of murphy
The concentration of catalase may change in different murphies which are stored in different ways before experiment. Storage at high temperature may do the enzyme to denature
Part of murphy used
Different parts on the murphy may hold different sum of catalase.
The consequence of temperature on the enzyme activity
The consequence of different concentrations of substrate on the enzyme activity
The consequence of pH on the enzyme activity
The consequence of concentrations of enzyme on activity of other type of enzyme such as amylase on amylum
The consequence on the rate of reaction of H peroxide by utilizing different concentration of Fungi as the beginning of catalase