If you through a ball twice and caught it there would be a higher chance of dropping it if you were only to do it once. Using the same logic the probability of molecular increases because of their speed they will occupy more space in the amount of time. The rate of an enzyme catalysed reaction may be measured either by the rate at which product is formed or the rate at which substrate is used. The effect of any factor is measured when all other factors are optimum and at time = 0. Rate is directly proportional to the enzyme concentration (if substrate maintained). This is because the rate of doing work is equal to the force multiplied by the velocity. The rate increases with increasing concentration of enzyme until all active sites saturated.
Temperature, rate doubles for a 10-degree rise. Above optimum protein denatured.
Inhibition
Reversible - if competitive the inhibitor is structurally similar to the substrate and competes for. The active site - If non-competitive will connect away from the active site but still affect it.
Because of these two things, there is more chance of an enzyme-substrate complex forming whether through actually colliding, or causing a collision.
It is reasonable to believe that an optimum temperature in catalase, an enzyme found in mammals, is the common body temperature of 36ºC. However, there are many types of catalase, and the variety in liver may be different as it grows, and has evolved in a different habitat to mammals. Some enzymes even have much higher optimum temperatures, for example bacteria that reproduce in very hot places.
We know if the temperature is raised too high, the enzyme will denature as high temperature produces excessive vibrations and cannot sustain life, which disrupts the precise 3-D structure and breaks the hydrogen bonds. It is reasonable to believe that this will happen shortly after 36º C, so I make it around 46º C.
If the enzyme were cooled to a point the enzyme will not work at all. I cannot predict the exact temperature but can be sure it is no higher that 10º C, I will not be able to prove this as I don’t have enough time. The lack of energy is the cause of the enzyme to stop working.
Predictions
Until denaturing the rate will rise as the temperature does.
The optimum temperature of catalase will be 46° C.
Denaturing will occur below 50º C around 47º C.
The Q10 value will be 4.
There are many variables but several ways to stop them. They include:
pH – pH is the concentration of hydrogen ions in a solution. If the pH is lower this indicates a higher concentration of hydrogen ions. Changes in pH will also denature the enzyme by changing the shape of the enzyme. pH must be synchronized carefully as it would affect the rate of an experiment. The regulation should be for the optimum pH, as a pH different from the optimum can denature the enzyme. The majority of enzymes work at their peak at a pH of 7, and this is true also of catalase. Enzymes are also modified to work at a specific pH or pH range. Light – Under aerobic conditions, sunlight can inactivate catalase. As the production of oxygen allows aerobic condition, the experiment, and liver should be kept out of direct sunlight. Enzyme Inhibition – This is where an enzyme is rendered powerless to be used to catalyse a substrate, as the active site is deformed, denatured, or occupied by another molecule. Meaning the "key" no longer fits the "lock". Non-competitive inhibition is permanent, while competitive is reversible, however, both are undesirable in the experiment. Catalase can be inhibited by ascorbate, ammonia and Cu2+ ions amongst others. as long as the apparatus is washed with distilled water and or sterilised such enzyme inhibition will not occur. Allosteric Interactions may allow an enzyme to be temporarily inactivated. Binding of an allosteric effecter changes the shape of the enzyme, inactivating it while the effectors is still bound. Noncompetitive Inhibition occurs when the inhibitory chemical, which does not have to resemble the substrate, binds to the enzyme other than at the active site. Competitive Inhibition works by the competition of the regulatory compound and substrate for the binding site
Volume and Concentration of H2O2 – This is always kept constant, as this will totally change all results that we have collected, because excessive number of hydrogen peroxide molecules will meet enzyme active sites quicker than a lower concentration. For a fair result the stream of catalysis must be constant and there must be an excess of enzyme to catalyse the substrate. The amount of liver to be used will be decide with test experiments that will allow me to see how the experiment works and what weights and concentrations to be used. Concentration of Enzymes – Reactions are created when enzyme molecules collide with substrate molecules. The more molecules that are in the solution the higher the chance that contact will be made. I will have to use the same mass of liver every time; this is not accurate because there may be different amounts catalase molecules in the same mass of liver. Different liver samples may contain different quantities of catalase, it is better to use always the same piece of liver. I may not be able to do this due to the fact of the liver is cut up and identifying each piece is not possible. I will decide on how much I will use using the test experiments. I will also have to use the same amount of catalase solution. Surface Area of Liver – just using the same amount of liver every time is not completely precise so I will use a mortar and pestle to grind the liver down into a pulp so the surface area is the same every time, I made need to use sand to help, to cause more friction. I will use sand because it doesn’t react with anything I am using in the experiment. Atmosphere and Pressure – This should be kept constant with out any oxygen to stop sunlight affecting the results but I cannot record or change the atmosphere or pressure surrounding my experiment
Temperature of the Substrate – This will be kept constant using a water bath and a thermometer. However, when cold liver is added it may cool the heated H2O2.
Implementing
I shall be using the following materials and apparatus to set up my experiment correctly and efficiently:
Weighing scales (correct to 1 decimal place)
Burette
500ml beaker
Heat resistant mat
Clamp
Clamp stand
Liver
Test tube with a glass tube coming from the side of it
Water bath
Scalpel
Tweezers
Thermometer
Stopwatch
0.5 concentration of hydrogen peroxide
Distilled water
Kettle
Cutting mat
10cm3 measuring tube
The variables below will have to be decided upon depending on the test experiments and to get the best results from the experiment. I need precise and correct results to create accurate graphs and charts so the experiment cannot take excessive time otherwise we will not have time to collect all the results not making the graph accurate and show of overall results, but I needn’t be too concerned with that as catalase is renowned as one of the quickest catalysts and enzymes in the world. The variables that I need to consider and change include:
Mass of liver: I will have to make a test experiment and this will tell me how much liver I need to use if I use to much I will not be able to be recorded as the Bunsen only holds 50cm3 of gas and will make more gas that it can hold. But I if don’t use enough I will not get results I can make good results from. The mass of liver must remain constant through all the experiments. I can only use the weight of the liver as a measurement because unfortunately the surface area cannot be calculated, as liver is not solid and cannot be cut into a certain shape. If I use too much hydrogen peroxide would be a waste and if I use too little all the enzymes will not be used.
Test experiments: In these small investigations, I am not looking for absolute accuracy, I shall be investigating simple measurements but they are that are critical to the real practical. The experiments I will perform will be according to the same procedure as I have planned for the real experiment and diagrams below but at room temperature around 30° C. it is no where near the point denaturing and not close to halting the enzyme.
Safety
Hydrogen Peroxide is an oxidising agent and type of bleach, eye protection such as goggles and gloves to protect the hands must be worn, the concentrations I am using are not extremely harmful but precaution must still be taken. If the hydrogen peroxide made contact with the eyes, skin, or clothes, the contaminated clothing must be removed, the skin or eye flooded with distilled water, a medical kit is needed to be at hand in the lab. Medical advice would be necessary if there was any pain caused or any marking of the skin. The H2O2 when reacting has a tendency to froth, care should be taken with regard to the amount of hydrogen peroxide used not to spill or expand over the volume of the test tube or beaker. If consumption of H2O2 much water and milk should be drunk and medical is advised
Bunsen burners should be left on yellow flame whilst not being used as they are difficult to see while on the blue flame and it isn’t as hot as the yellow flame. The Bunsen should be moved by the edge of the base, as the metal tube will be hot. If burnt for any reason cold water should be applied to the effected area. If glassware is broken it should be cleared by using a dustpan and brush no human contact should be made with the broken pieces. Exits should be clear, and there should be more than one visible. Unused stools and bags should be placed to one side in case of emergency in the lab. Lab coats should also be worn in case of spillage. This avoids uniform and exposed arms being injured.
I shall proceed with the experiment but with great detail to all tasks I will performed; I shall begin by washing out all the apparatus with distilled water and place all apparatus I shall need out and have a check list. I shall have to cut each piece of liver each time I need it other wise it will become dry and affect the results. The liver cannot be emulsified due to the difficulty of collecting after it has been and the use of sand was impossible, due to collecting the weight of the sand afterward would not been possible. The kettle shall need to of been boiled to 100oC and cold water at hand to get it down to the correct temperature.
The other apparatus should be set up like follows: the test tube with the glass tubing coming out of the side of it to the water bath, with the glass tube end inserted 1cm into the overturned burette filled with water, the water is reading 50cm at the top. A note should be taken if the water level is below 50cm.
I will use the kettle and cold water to get these temperatures, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C and 80°C which is also subject to change after a period of time. The test tube should be placed in the 500mm beaker for at least 3 minutes to warm up the surrounding water - acclimatise. We can place a thermometer into the catalase solution but it might carry some of the fluid out when removed, so I decided not to. So one will be suspended next to the test tube
The experiment shall have to be carried out as follows: Start the kettle boiling, then fill the water bath and burette with water, be sure to have the bottom of the burette open. Turn the burette over and place your thumb over the hole to stop the water from escaping. Place the bottom of the burette into the water bath so the hole is submerged in the water. To get the water level down to the 50cm mark you must open the top of the burette until enough water escapes. Then collect 10cm3 of H2O2 and pour it into the test tube. Place the test tube into the beaker and pour the boiling water into the beaker leaving enough space to supplement it with cold water, to bring the temperature down. Once the correct temperature is reached (let it be a few degrees above because it will cool. Then leave the test tube in the beaker. Make sure the glass tube protruding from the test tube is securely placed under the hole of the burette. Measure out 1 gram of liver using scalpel and tweezers, placing it on to the weighing scales with care. Once the liver is ready check if the temperature is still correct adjust it if it isn’t. place the stopwatch close by ready to press the start button. As soon as the liver is placed into the test tube push in the bung and start the stopwatch. Keep an eye on the experiment at all times and keep notes of lots of times near the end were it is close it stopping as this may affect your results if you leave it too long.
Here is how my apparatus is going to be set-up:
The first experiment is at room temperature but once the other temperatures are reached, buy collecting the boiled water and supplementing it with cold water. The test tube can remain in the water bath as the experiment is completed. The H2O2 is already in the test tube and the liver must be placed in quickly. The boiling tube shall be resting on the side of the beaker place it in the groove on the lip to keep it secure. Place the liver into the test tube and quickly place the bung onto. Start the stopwatch as soon as the bung has been placed on top. Once it has stopped creating gas, which will be around 10cm3, record the time to one decimal place. The apparatus must be cleaned ready for the next experiment. The experiments should be repeated twice each.
Results
I shall create a table where I can put all my findings in a clear and precise fashion. As well as having final results on it. The final results table will look like this:
Due to time restraints, I shall do very quick test practicals and this will allow me to decide on the measurements of the catalase and amount of liver to use. I first started off with a concentration of 2 molar and 5g of liver with excess H2O2 but this went off the scale and had to start again. So, I used 2g and 1 molar with excess H2O2, but it still went over the scale but not as fast so I used 1 molar and 1 gram and it made about 40cm3.
Results
Analysis of Results
From the graph, it is clear that up until 55°C the rate of the reaction increases. After this temperature, which can be assumed to be the optimum for catalase, the rate decreases. The rate begins to decrease at 56°C. As temperatures get higher than this the enzyme starts to decompose and not catalyse. However, at 80°C there is still slight action the investigation I did proves this but I had to stop as the enzymes stopped working after about 10 minutes and no more bubbles were produced. The reaction caused the H2O2 to bubble up and create froth in every experiment but soon cleared before the reaction had finished. There is a simple straight-line formation in all the results apart from the 80°C. This suggests that changes in temperature are directly proportional to the enzymes.
Conclusion
When I predicted that the rate increased with the temperature I was correct. I decided this upon some findings; heating, or providing kinetic energy to the molecules make them cover more space in the same time. The quicker a particle moves, the more collisions it will make it reacted with all of the other molecules faster with an increased probability of reacting due to a greater force of collision when they do collide. The optimum temperature was predicted as around 46°C and was incorrect but fairly close. There was evidence it could be as high as 80°C. Its optimum temperature as predicted isn’t in cold conditions but at fairly temperature. Scientists say that contact made with a substrate molecule is enough for catalysis to exist. This would clarify the high rates at lower temperatures. There is a difference in rate between different temperatures. At room temperatures the rate of catalysation was high, so in lower temps it would also work well like underground in potatoes. It is known that small rises in temperature can make a change to the energy the particles have, but it is known that the amount of gas does not necessarily increase. This would explain why 30°C and 35°C are similar in their reaction rate.
The rate beings to slow once the optimum point of rate has been reached after that the enzyme begins to decompose and fail to perform at there full potential. This suggests that above 56°C, the vibration caused by high kinetic energy is so high it disrupts the delicate 3-D structure. This bring me back to the hypothesis with the non-competitive inhibition and competitive Inhibition. Adding more substrate reduces inhibition. The combination is reversible.
This investigation was reliant on the mass and temperature of the enzymes. But using my method creates several problems. The liver was always used at room temperature. Not only is this an inconsistent value due to doing the experiments over a series of days, adding cold liver to warm H2O2 cools the enzyme. As you increase the temperature the percentage of error increases. The fact that the liver was not warm proves the theory of kinetics is true the fact that it may have been cooled affects the results but not the conclusion. Also when the bung was press into the test tube it displaced some of the air inside the tube and made the results different and also it took time for the bung to be placed on to f the test tube the reaction had already started to take place and this too would of affected the results. The enzyme showed signs of denaturing at 57°C, but did not denature fully until around 80°C. The outer cells containing catalase may of reached the denaturing temperature but not the coated ones.
How much enzyme is in 1 gram of liver is questionable. The same weight of live doesn’t truly determine the same amount of enzyme contained in the liver. So it a piece of liver was collected from the centre of the liver it may contain more or less enzyme than the edge or vice verse or maybe the liver contains the same amount all over. The liver was kept over a series of days and this would of affected the amount of water in the liver. If the water evaporated the catalase would be in a dehydrated form then there could be more in a dehydrated form meaning there for the weight would remain the same but the amount of catalase could be increased. To over come this problem you could dehydrate all of the liver or keep it all moist.
I found/collected information on how the errors could be calculated to a percentage this is what I found:
To calculate the maximum percentage error, take the minimum value measured by this piece of apparatus.
- Burette has an error of ± 0.04ml. Minimum quantity used for was 5ml.
±0.04/5 = ±0.8%
- Thermometer has an error of ± 0.5°C. Minimum quantity used for was 15°C.
±0.5/15 = ±31/3%
- Measure has an error of ± 0.01g. Minimum quantity it was used for was 5.22g.
±0.01/5.22 = ±0.19%
- Time measurements were taken to the nearest second. Minimum amount of time was 110 seconds.
±0.5/110 = ± 0.455%
- measuring cylinder had measurements correct to ±0.05ml. All cylinders volumes were 10ml.
±0.05/5 = ±1%
Total: ± 1 ± 0.455 ± 0.19 ± 31/3 ± 0.8 = ±5.78%
Thus the shortest amount of time (110 seconds) could be 104 – 116 seconds.
