Non active site inhibition
This is simply the fact that inorganic ions like Na or H can distort the shape of enzymes by binding to allosteric cites which are a kind of activation site away from the main active site thus changing the globular structure and in turn the shape of the active site so no substrate may enter or fit.
Another type of non active site directed inhibition is when an ion just simply binds to a part of the enzyme and changes its shape so it is denatured when there is no active site.
Both of these may happen in my experiment on the affects of Ph on catalase but I am inclined to think that the amphoteric affect is the most likely.
Immobilised enzymes
These are when enzymes processed so that they can be reused commercially to save costs, they can be added and removed easily, can be used in continuous process, and are more stable against heat and increases in temp. There are two ways of immobilising enzymes. Trapping them in beads or in membranes. Mainly alginates are used to trap enzymes in beads and resins in membranes.
You can make immobilised enzymes by mixing an enzyme with a solution of sodium alginate and then using a syringe pushing drips of the solution into a solution of calcium chloride. This creates porous beads of sodium alginate containing the enzyme.
Catalase
Catalase is an enzyme that speeds up the break down of hydrogen peroxide to form water and oxygen. Catalase has very low activation energy of 86 kJ mol. It works best at pH 7 which is neutral and at about 37- 40 degrees so it s a mesophile meaning that it works best at temperatures between 20 and 40 degrees.
Good reaction situation:
If Enzyme Becomes Denatured:
Preliminary results
I did preliminary results to find out how my experiments may go.
2grams of liquefied potato
Buffers A and B stand for citric acid and anhydrous disodium hydrogen phosphate.
2grams of liquefied potato
Buffers A and B stand for citric acid and anhydrous disodium hydrogen phosphate.
Plan
Potato-needed as contains catalase
Citric acid-needed for buffer
Anhydrous disodium phosphate- buffer
Beaker-to hold solutions
Grated cylinder-measure oxygen displaced
Plastic pipe-conduct water to the grated cylinder
Hydrogen peroxide 10 VOL-be broken down by catalase
Syringe-measure hydrogen peroxide, hydrochloric acid, buffers sodium hydroxide.
32 % Hydrochloric acid- to create a ph of two for preliminary
Sodium hydroxide- create a ph of 13 for preliminary
Conical flask-to hold reacting products
2 hole bung- for syringe and plastic tuning
Scales-to weigh amount of potatoes used
Blender-to blend potato
Sodium alginate- create beads
Calcium chloride- to form the beads in which the enzymes are
Diagram
Risk assessments
There are several risks that will be involved in this experiment, for I am using hydrogen peroxide which is very corrosive so I will have to handle with care and use gloves. My hydrogen peroxide is broken down quickly in UV light so I will have to store it in dark places. The same goes for hydrochloric acid, as I will be using 10 vol so its concentration will be very high and I will have to use protective gloves and goggles. I will also use special bottles to hold the hydrogen peroxide and hydrochloric acid that has a non-slip grip surface even when wet so chances of dropping any bottles are severely reduced.
Hydrogen peroxide:
It is an oxidizer, which is not flammable but may start a fire if put in contact with organic materials, so keep the area well ventilated. The maximum concentration that is safe to use in the laboratory is 20 vol. Make sure eye protection and protective clothing is worn, as hydrogen peroxide is corrosive. In case of any solution getting in contact with the eyes, flush it out with copious amounts of water and seek medical assistance. Any contact with the skin should be avoided but in case there is any contact, wash it off immediately with plenty of water as burns may result. Spillages should be diluted down with lots of water and washed up immediately. Hydrogen peroxide should be stored in a clearly labelled dark bottle, as it decomposes quickly with the presence of light, in a cool place.
PH buffer solutions:
As the solutions are of various pH between 4.4 to 7.5, they are acidic and basic, so solutions can have corrosive or caustic properties. Wear eye protection and avoid contact with the skin. If there is any spillages, including on the skin or eyes, wash it off immediately with plenty of water, especially if any of the solutions come into contact with the eyes. Store the buffers in clearly labelled bottles.
Glassware:
The syringe plunger slides out easily, therefore secure it to the main part of the gas syringe, but also make sure that this will not restrict any movement of the plunger.
Take care of other glassware, keeping them away from the edge of the workbench to avoid any breakages that may cause injury, especially the boiling tubes as they can roll around.
Potato: It is of low risk, but should be kept in a clearly labelled container.
Products of reaction: The product is water so there is a low risk. However, as the pH of the buffers present in the mixture of the reaction, in some experiments, have slight hazards, avoid contact with the skin. To dispose of the residue, decant off any liquid present, washing it away down the sink and use a spatula to remove the potato out of the boiling tube to be discarded.
Control
Since in this experiment I am varying the pH of the solutions, the state in which the enzyme catalase is in whether it be free or immobilised I will need to keep a constant by which I may be able to accurately and surely record my results. The constant that I shall be keeping the same in my experiment thought by which I may measure everything by shall be the mass of the potato which shall be at a constant 10grams plus or minus 0.10g.
I shall also be controlling the hydrogen peroxide solution to a 10-vol solution of 5cm3. The temperature shall also be kept constant at a temperature of 40 degrees, as this is roughly the optimum temp for the enzymes to break down the hydrogen peroxide. This is due to the enzymes being mesophiles (optimum temp of reaction below 40 degrees). To control the temp I shall be using an electronically maintained water bath, and a thermometer to check the set temp.
I shall also be controlling the bead size as this may slightly affect the rates of reaction so the same size nozzle shall be used, this will be cleaned regularly Incas of clogging which may upset the bead size and create possible anomalies.
Finally rigorous measuring at stages so as not to upset the ph balance thus affecting the results of the experiment will control the ph solutions.
Method
Before I started my experiments I made two stock solutions of 250cm3 one with5.25g of citric acid (A) and the other with 7g of anhydrous disodium phosphate (B). I then created different ph solutions; the table below will explain in detail how to create the different ph solutions.
The above table shows the ratio to which I mixed the solutions A and B to make the variable ph contents.
To obtain the enzyme catalase I bought some potatoes roughly weighing 500 grams each. I then skinned them, chopped them up and liquefied them in a blender. I measured out roughly 10 grams of liquefied potatoe plus or minus 0.10grams, as that was the greatest degree of accuracy that the scales I was using could offer. I measured out 10 grams about 30 times as I would need 10g 5 times for each experiment with different pH, I would be doing 3 experiments for immobilised and free enzymes.
Now I needed to create my alginate solution from which I could make my beads and thus create the immobilised enzymes. I did this by mixing 8cm3 of distilled water with 2 grams of sodium alginate but the alginate was added to the water while it was being heated in a water bath while being stirred gently so as not to make it too thick.
After that I created my calcium chloride solution by mixing 98cm3 with 2 grams of calcium chloride this was mixed thoroughly to make a very evenly distributed.
Next I needed to create my immobilised enzymes and I did this by weighing the alginate solution and then adding 5 grams of potato 15 times. Then I poured the solution into a syringe and added drops to some calcium chloride. I did this till all the alginate potato solution was gone. Because I had bothered to weigh my alginate solution before I was able to extrapolated the mass of beads I needed that would have exactly 5 grams of potato to 0.10 grams.
I obtained my 5cm3 hydrogen peroxide solution by using a syringe to remove it from a beaker to which I had originally poured it into. Before this I measured out 300cm3 of 10-vol hydrogen peroxide, as this would be the exact amount needed for all of my experiments using free and immobilised.
With the measuring of the hydrogen peroxide it marks the end of my pre set ups and allows me to actually set up my experiment as above in the diagram.
For the experiment I got a conical flask and put the right ph concentration in it and the potato purée in whether it be immobilised or not. Then I put a2 hole bung on the top, with one hole holding a pipe that was connected to an upturned grated cylinder full of water partially submerged in a tank of water.
The other hole had a testtube in it that had exactly 5cm3 of 10-vol hydrogen peroxide. I then injected the hydrogen peroxide into the conical flask and started a stopwatch at the same time and after 30 sec and 60 sec I would take results and then waited to the very end to take the overall results. I did this three times for immobilised and non-immobilised catalase. From these results I was able to find out whether immobilised enzymes were more or less affected by pH and draw graphs.
Results tables
Test 1
Test 2
Test 3
Averages
Analysis
After analysing the data from the experiments and tabulating them I have concluded that immobilised enzymes when compared to free enzymes are less affected by sudden changes in Ph. And I have also proved my hypothesis right on how the volume of oxygen would be greatest at pH 7. And the overall amount of oxygen production would be greatest in immobilised enzymes. This could be due to the fact that the actual immobilising of the enzymes help provide a protective barrier around the enzymes not allowing the H+ and –OH to act as inorganic ions and attaching themselves to the enzymes thus distorting the active site. Also the immobilised enzymes provide a barrier from the pH so the enzymes do not have to take up or donate H+ so this reduces the amount of disabled ionic bonds providing a more stable enzymes that are able to carry out the reaction of breaking down hydrogen peroxide.
This can be proved by looking at the average volume of oxygen produced at different pH with free and immobilised enzymes, the results show that on average the free enzyme results are much lower. An example is the results at pH 4.4 and 7.5 where with free enzymes at 4.4- 29.2cm3 of oxygen was produced and at 7.5-44.3cm3. This compared to immobilised enzymes where at 4.4-35.7 cm3 of oxygen was produced and at Ph 7.5-46 cm3 was produced.
To analyse my results further I drew a table that had the sum of the average oxygen production for immobilised and free enzymes, this table showed the clear difference of oxygen production of a8.8 c3 as immobilised enzymes produced 231cm3 while free enzymes produced 212.2 cm3 this has again further help me conclude that pH has less of an affect on immobilised enzymes. These results me that catalase have an optimum pH of 7, which is neutral, and works best between pH 6. And 7.5 as between these ranges the volume of oxygen showed a significant increase. All these results showed that catalase obviously has its active site denatured by pH due to the pH as the ionic bonds are weakened due to the carboxyl group giving up hydrogen atoms in alkaline and the amine group taking up hydrogen atoms in acidic conditions. The results have also drawn me to another conclusion one that was not apparent to me before. The conclusion is that the more acidic or more alkaline the conditions the less oxygen produced, less hydrogen peroxide broken down and thus the less active sites available to break down substrate. This is because in not so acidic or alkaline #e conditions like Ph 6 and 8 their would be more oxygen than at 5 and 9 this is due to the concentration oh hydroxide and hydrogen ions in the solution. What is meant by that is that the stronger the more extreme the pH the more hydrogen the enzyme gives up or takes so the more ionic bonds weakened or broken. This in turn means that in pH closer to neutral the less destabilising of enzymes and less distorting and denaturing of active sites some more oxygen is produced. This is unlike more extreme pHs like 1 and 14 where a lot more hydrogen has to be take n or given up to try and neutralise the solution and so more distorting of active sites, due to destabilised ionic bonds. Analysing the graphs of the average oxygen produced at different pH it further helps my conclusion on how immobilised enzymes have a greater tolerance for pH than free enzymes.
Evaluation
The conclusion can now be stated with confidence as the results and theories produced are substantial for accurate and reliable conclusions. But these conclusions are based on the accuracy of the results I had gathered which may have been in correlation with the hypothesis and all of my research the actual way I investigated could have been greatly improved in accuracy.
For instance if I was able to do this experiment again I would use catalase from the liver as it has been suggested that it would give a greater yield of catalase and so would produce more oxygen from the same arbitrary units of catalase making my results a lot easier to read and put down.
I would also use an electronic scale, which had a greater degree of accuracy in terms of decimal places. This is due to the fact the scale only went up to 2 decimal places and even then I weighed the mass of potatoes to plus or minus o.10 grams and next time would use a more accurate scale and try to be more accurate in weighing the potatoes myself.
Also if I were to redo this experiment I would dry the potato so it would provide more accurate results, as the water would not be weighed but jut the dry mass of the potato.
I would also use a pH meter to test the acidity or alkalinity of the pH to make sure I had exactly a pH of 4.4 or 5.3 as I might have measured out the wrong volume of each buffer. This brings to me another point which concerns the actual volume of solutions A and B that I used, to make my results more reliable I would either use a smaller scale measuring cylinder to measure the volume. Or I could work out how much one mole of solution A and B weighs in grams and then from that I can weigh the solution on a scale to see what mass of each solution should be used. Another part of my experiment that I could improve was the actual size of my beads as I feel that I should have made them all the same size as I only made them by squeezing the alginate the solution through a syringe. So next time I would use the proper apparatus that would make beads of exactly the same size, concentration of catalase and hopefully the same mass.
When collecting my oxygen the pipes I used to transfer the oxygen to the unturned grated cylinder were not insulated with Vaseline to produce an air tight passage which could have resulted in losses of oxygen and rendered my results anomalous and useless but which when looking at the results table doesn’t seem to have occurred.
Whether or not I did this precise experimental procedure again, if I were to do one which included the use or an alginate solution n I would use a magnetic mixer as it would mix the solution quicker, make it smoother and save me stirring the solution with difficulty in whilst it resides in a water bath, which could be potentially dangerous.