How does pH affect the Denaturation of enzymes Starch and Amylase.
How does pH affect the Denaturation of enzymes
Starch and Amylase
Hypothesis
A Ph too high or too low for the amylase will slow down and denature the enzyme.
Background Information
Starch
Starch consists of two polysaccharides amylose and amylopectin. The two polysaccharides are made of glucose monomers. Monosaccharides are sugars formed by the different formation of Carbon, Hydrogen and Oxygen. The most common Monosaccharide is glucose. Two glucose molecules join together through a condensation reaction (the loss of a water molecule) and become a Disaccaharide. The link between the two is called a glycosidic bond. Monosaccharides can also become very large molecules filled with lots of sugar molecules joined again by glycosidic bonds and become polysaccharides. The test for Starch is to add iodine, which turns the starch solution blue/black. If the starch is broken down and iodine is added it would not be blue/black as no starch would be present.
Amylose
Amylose is a linear polymer of glucose joined by ? (1>4) linkages. 1>4 refers to the linking of carbon atom 1 of one monosaccaharide and carbon atom 4 of another, and between carbon atom 1 and 6 on a 1>6 link. The number of glucose units in amylose changes from sample to sample. Generally there is over 300 glucose units per amylose molecule. It is described as linear as it as an unbranched helical chain. Fig. 1
Amylopectin has a much greater molecular mass than amylose. It is a branched chain and has over 1300 glucose units per molecule. This structure contains (1-4) linkages (as amylose) but with a greater proportion of (1-6) linkages, which gives a large highly branched structure. Fig 2
Amylase
Amylase exists in many forms and plants and humans make use of it. There are two kinds of amylase enzymes. Alpha-amylase is found in saliva and is called ptyalin. The other kind is called pancreatic amylase and is secreted in pancreatic juice, into the small intestine or ileum.
How enzymes work - 'The lock and key theory'
Enzymes work as catalysts by combining with a substance known as the substrate, to form the enzyme substrate complex. This then breaks down releasing the enzyme and the product. Amylase is an enzyme, which works as a catalyst. This means that it is unchanged by the reaction, but makes the reaction easier by reducing the energy required for it to happen. Catalysts speed up the reaction. The reasoning behind the working is called the "lock and key" theory. Every enzyme is specifically shaped so that the products can fit into them perfect. When they are in place they react, and are released to be used again. Starch is broken down by hydrolysis. Hydrolysis breaks down starch by the adding of a water molecule. So starch plus water becomes maltose.
The activation Level from catalysts
In order for colliding particles to react together, they must have enough energy to exceed the activation energy needed for the reaction.
The catalyst provides a different route for the reaction, a route with lower activation energy. This means that more particles in the reaction mixture have sufficient energy to react. It can be compared to lowering the bar in a high jump competition: a lot more people can get over the bar at a lower height.
Denatured
This means that the enzyme has lost its ability to function as a biological catalyst. Its active site has changed shape preventing the substrate binding to it. This can occur because of high temperature or change in pH.
Factors affecting enzyme activity
. pH
Enzyme activity is high only within a narrow range of pH. Outside this range the activity of this enzyme falls quite rapidly. This is because the enzyme loses its shape i.e. it is denatured. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. In general enzyme have a pH optimum. However the optimum is not the same for each enzyme.
2. Enzyme concentration
The rate of enzyme activity increases as the enzyme concentration increases (provided that the substrate concentration is not limiting).
3. Substrate concentration
The rate of enzyme activity increases as the substrate concentration increases. When all the active sites are occupied, increasing the substrate concentration will no longer increase the rate - substrate saturated.
4. Temperature
The rate of chemical reactions increase as temperature increases. Human enzymes work best at 37 degrees Celsius while most plant enzymes work best at about 30 degrees Celsius. These temperatures are called the optimum temperatures. Above these temperatures the enzymes start to denature.
Inhibitors
Inhibitors shall not affect my experiment but the rate of enzyme-affected reactions may be decreased by the presence of inhibitors. There are two types of inhibitors: Reversible and non-reversible inhibitors.
Risk assessment- COSHH
Starch - Very low risk. Ensure that it is kept in a clearly marked containe
Alpha-amylase- In powder form it is an irritant to skin. Wash thoroughly with warm water if contact occurs
Iodine- Can be toxic and an irritant. Eye protection should be worn. Any spillage onto the skin should be washed away with water. Only small drops should be used.
pH Buffer Very low risk.
Method
The method I am going to use to prove my theory is to time how long it takes for amylase to break down starch. ...
This is a preview of the whole essay
Starch - Very low risk. Ensure that it is kept in a clearly marked containe
Alpha-amylase- In powder form it is an irritant to skin. Wash thoroughly with warm water if contact occurs
Iodine- Can be toxic and an irritant. Eye protection should be worn. Any spillage onto the skin should be washed away with water. Only small drops should be used.
pH Buffer Very low risk.
Method
The method I am going to use to prove my theory is to time how long it takes for amylase to break down starch. To make it relevant to how pH affects the rate of reaction, I will have solutions made up from pH 3, up to pH 11, four levels higher and lower than the neutral level. In a boiling tube I will have 5ml of starch, and I'll add 1 ml of iodine. The starch should turn black/blue to show that Starch is present. Then I will add a solution of a 5ml of the variable pH. Finally I will add 5ml of amylase LAST, as this will be when I will need to start the stopwatch. I will put my thumb over the tube end and give each one a gentle shake to mix in the different solutions. To show that the amylase has broken down into maltose, the solution should turn colourless - this will be when I stop the stopwatch. To match up with what colour I want the solution to be at the end, I shall set up a boiling tube which has already had starch broken down by amylase, so I have something to compare with.
The variable that I will have will only be the pH. Every other factor, the volumes, concentrations and temperature, (presuming that the room always stays the same temperature) shall stay the same. Obviously the time shall change only as a result of the varying length in rate of reaction
Apparatus
9 boiling tubes
pH sensor
Measuring cylinder
50 ml 1% starch
50ml 1% amylase
Iodine solution
Pipette
Stopwatch
Pilot study
A pilot study will be carried out to ensure that the correct measurements of solutions and time span are suitable for my experiment. Only one set of results shall be recorded as the reliability of having more than one set of data is not crucial for my pilot study.
The method I will use for the pilot study will be to have 5ml of a set pH put into a 30ml boiling tube. I shall then use a pH strip to test that the pH is at the correct level. Then I shall add 5ml of starch to the solution. I will add 2 drops of iodine into each boiling tube and give each tube a gentle shake. The solution should turn blue/black to show the presence of starch. I will then add 5ml of Amylase to the solution As soon as I add the alpha-amylase I will start my stopwatch to time how long it takes to go colourless. This will show that the starch has been broken down into maltose, leaving no starch present.
Pilot study results
Ph
Time (s)
Observations
2
No change
Did not change after 30 minutes
4
343
Took a while to change
6.4
65
7
05
9
62
2
2
Turned colourless straight away
I found out that the results produced came out in a reasonable time scale, and did not happen to slow or too quickly. For my actual test I will now keep the same proportions and measurements of solutions the same, and I will not introduce any modifications on percentages or volumes for my full experiment. The pH range I asked for was to between 2 - 11, and I think the range I was give will be suitable for my final experiment. What came out, as an anomalous result that was unexpected from my research, was that pH 12 reacted and turned colourless almost straight away. So I set up another tube of pH 12 at 5ml, and added 5ml starch. I added the 5ml of amylase and gently stirred it. I was going to test if I needed to add more drops of iodine to make the concentration of iodine stronger in the solution. I added 3 drops this time and the same thing happened in that the solution turned colourless straight away. I was sure that this should not be happening so I set up the experiment again, except I didn't add any amylase. I was going to add the amylase after I had put in the 3 iodine drops and see if this mattered. As I put the iodine into the ph 12 and starch, it turned colourless straight away. This confused me, as what should be breaking down the starch was amylase, and as no amylase was present something else must have been affecting my results. I tried another experiment with just pH 12, and added drops of iodine to it. The pH turned colourless straight away, showing that it was the high pH 12 that was making the iodine colourless, and no other factor. To query about the other levels of pH, I set-up tubes of all the other pH levels and added one drop of iodine to each, waiting to see if any other change occurred with just iodine added. After 20 minutes of observation, no change happened, assuring me that no factor would be changing my results in my final experiment. This meant that only pH 12 was causing a reaction with the iodine, and something in the pH 12 buffer reacts with iodine to be colourless.
Apart from my findings on PH 12 effects, I shall keep everything the same as my pilot study with volume and proportions, and still only add 1 drop of iodine. I will also still use pH 12, as I have no control over the pH given to me. The only change I shall make is that I will have a water bath set-up at 40?C. This will suit the enzyme as it works best at around 37?C, and shall not affect my research on the effects of pH, as all boiling tubes will be placed in the water bath.
I also decided to help me know when I shall stop the stopwatch; I will set up a tube of Starch and Amylase mixed, and add a drop of iodine. I will then allow it to become clear. I will do this before I start my experiment and I shall use this next to the solutions I'll be watching to determine for me an end point, so I know when to stop my stopwatch, as it was sometimes difficult on the solutions that took longer to change in my pilot study.
Method
From deciding in my pilot study, before my experiment began, I set out a boiling tube with 5ml of starch and 5ml of alpha-amylase mixed. I added one drop of iodine and waited how for it to turn completely colourless. The time it took for this to happen did not matter as this will be an end point which I will compare with whenever I want to see what the colour change should look like at its final finishing point.
To begin the experiment I set up six 30ml boiling tubes. I marked each of these with what pH level was going into it. This was to be pH levels 2, 4, 6.4, 7, 9 and 12. Then in the rack I added the corresponding pH level to what I wrote on each boiling tube. To check the pH was what I was told it would be, I took a strip of pH paper and tested the bottle that each pH buffer came out from. Each pH level was as it was said to be and I carried on with my experiment. I then placed the boiling tube I was going to time in the water bath at 40?C, and added everything else to the boiling tube while it was placed inside the water bath.
I then carried the next stage of my experiment one step at a time. I used a syringe to correctly get 5ml of 1% starch volume from its container into one boiling tube at a time. I then placed 1 drop of iodine solution into the first level of pH in the boiling tubes. The solution turned blue/black in colour, showing that starch was present in the solution. I placed a rubber bung over the top of the boiling tube and gently shook the solution together. I then removed the bung and got 5ml of 1% alpha-amylase ready to be added to the mixture by a syringe. As I squirted the amylase carefully into the boiling tube, I simultaneously started my stopwatch. I timed how long it took for the solution to become colourless. I knew when to stop the stopwatch as I compared it next to the measure I had done previously before the experiment. When it did match the colourless solution, it showed that all the starch had been converted to maltose and the reaction was complete. I recorded my results in my pre-drawn table. I then did the same for the remaining results. As I started on pH 2, I then went in turn up to pH 12.
Fully aware of what pH 12 I timed how long it took for the pH buffer 12 to react with only the iodine drop. I decided it would not be necessary to carry on using pH 12 with amylase added, and use only the timings I got from adding iodine to the buffer.
After I collected all my results for each six-pH level and had them recorded in my table, I repeated the exact same experiment to get more accurate results, using new clean boiling tubes to keep things fair. I then recorded this second set of results and put them in the next column on my table. Having two sets of results enables me to get a mean score for more reliability.
The boiling tube I used to compare with when the reaction was finished also acted as the control for my study, as no pH buffer was added to the solution.
The dependant variable to my investigation was the pH level added to the solution.
The independent variable was the seconds it took for starch to be converted to maltose, when it became colourless.
All my variables, Temperature, concentration of alpha amylase and Starch, drops of iodine were kept constant to make my experiment as fair as possible.
Results
Ph
st test
2nd test
Mean value
2
800+
800+
800+
4
257
271
264
6.4
36
18
27
7
96
16
06
9
47
51
49
2
2
2
2
Table 1.
My Table (Table 1.) shows how long it took in seconds in experiment 1 and 2, for the starch to be converted to maltose at each of the different levels of pH. The table also shows an average of the time worked out by calculating the mean from each set of results.
I produced a graph (fig.1) showing me the results of my average results. I also added a line of best fit to show the linear relationship. The yellow triangles indicate the average time for amylase to digest starch at each level of pH. The line of best fit shows that, as the pH got higher, the reaction time got lower as well.
As the first result for pH 2, did not react with the enzyme, there is no actual time unit, but as I stopped recording at 30 minutes, this is what I put on the chart. To see the other results more clearly I removed the results from pH 2, as they ruin the graph in order to compare results. (fig.2) Shows the results without the pH2 for easiness of comparing. Both these graphs can be seen full size as appendixes in my coursework later on the next pages.
Discussion
The results of my experiment support my hypothesis to an extent.
'Hypothesis'- A Ph too high or too low for the amylase will slow down and denature the enzyme. My results show that a pH too low denatures the enzyme, which is what happened at a pH of 2.Then, as the pH gets higher, the optimum pH level seems to be around pH 9. It could not be optimum at pH 12, as this could not be tested to due the chemicals that altered the iodine in the pH buffer. Those 2 results on my graph are ones that can be seen as an anomalous result.
My table and graph both show that as the pH level got to pH 9, it also reacted the quickest. Where it the amylase did not react with the starch was at pH 2, the most acidic level of pH I used. It did not react as the pH used would have altered the structure of hydrogen bonds in the alpha amylase, and therefore denatured the enzyme. PH levels are an important factor which enzymes require to be at the optimum for each condition for each enzyme to perform at it's quickest. Temperature, Substrate concentration, enzyme concentration are other factors which an enzyme requires to be precise for the enzyme to react quickest. The explanation for my results is that between the reaction of alpha amylase breaking down starch into maltose, the pH level that the reaction was happening in, has a direct effect on the outcome of the experiment.
Starch consists of two polysaccharides amylose and amylopectin. The two polysaccharides are made of glucose monomers. Monosaccharides are sugars formed by the different formation of Carbon, Hydrogen and Oxygen. The most common Monosaccharide is glucose. Two glucose molecules join together through a condensation reaction (the loss of a water molecule) and become a Disaccaharide. The link between the two is called a glycosidic bond. Monosaccharides can also become very large molecules filled with lots of sugar molecules joined again by glycosidic bonds and become polysaccharides. The test for Starch is to add iodine, which turns the starch solution blue/black. If the starch is broken down and iodine is added it would not be blue/black as no starch would be present.
Amylase is an enzyme, and acts as a catalyst. Amylase exists in many forms and plants and humans make use of it. There are two kinds of amylase enzymes. Alpha-amylase is found in saliva and is called ptyalin. The other kind is called pancreatic amylase and is secreted in pancreatic juice, into the small intestine or ileum. The reasoning behind the working is called the "lock and key" theory. Every enzyme is specifically shaped so that the products can fit into them perfect. When they are in place they react, and are released to be used again. Starch is broken down by hydrolysis. Amylase digests starch by catalysing hydrolysis. Hydrolysis breaks down starch by the adding of a water molecule. So starch plus water becomes maltose.
In my experiment this was what happened when no pH buffer was present and acting on my experiment. When a pH buffer was added, it could either speed up the reaction or slow it down, and as proved stop it reacting completely. Enzyme activity is high only within a narrow range of pH. Outside this range the activity of this enzyme falls quite rapidly. This is because the enzyme loses its shape i.e. it is denatured. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. In general enzyme have a pH optimum. However the optimum is not the same for each enzyme.
The two anomalous results I collected were from the same pH level this shows that it was not an error on my part, but something to do with the pH buffer at pH 12. What came out, as an anomalous result that was unexpected from my research, was that pH 12 reacted and turned colourless almost straight away. So I set up another tube of pH 12 at 5ml, and added 5ml starch. I added the 5ml of amylase and gently stirred it. I was going to test if I needed to add more drops of iodine to make the concentration of iodine stronger in the solution. I added 3 drops this time and the same thing happened in that the solution turned colourless straight away. I was sure that this should not be happening so I set up the experiment again, except I didn't add any amylase. I was going to add the amylase after I had put in the 3 iodine drops and see if this mattered. As I put the iodine into the ph 12 and starch, it turned colourless straight away. This confused me, as what should be breaking down the starch was amylase, and as no amylase was present something else must have been affecting my results. I tried another experiment with just pH 12, and added drops of iodine to it. The pH turned colourless straight away, showing that it was the high pH 12 that was making the iodine colourless, and no other factor. To query about the other levels of pH, I set-up tubes of all the other pH levels and added one drop of iodine to each, waiting to see if any other change occurred with just iodine added. After 20 minutes of observation, no change happened, assuring me that no factor would be changing my results in my final experiment. This meant that only pH 12 was causing a reaction with the iodine, and something in the pH 12 buffer reacts with iodine to be colourless.
The reliability of my results can be assessed by me using the results I collected. The results were fairly even on both times I did the experiment. If I had done anything major wrong I would have expected more anomalous results on my part, this was not the case.
My investigation was successful in supporting my hypothesis, but there are also some limitations that may have had an effect on my experiment. I kept the temperature constant by carrying out the experiment in a water bath at 40?C. This means that no results could be affected because of a change in temperature. My first limitation could be on deciding when the end point of my experiment. I only judged by comparing when I thought the experiment was finished, with an already reacted solution. Using a colorimeter can solve this problem. I would test an end point solution by putting it in the colorimeter and the amount of light passing through would be given out as a percentage. I would then only finish timing my experiment when the colorimeter said that the solution was at the exact same percentage of light transmission as my end point.
Another limitation to my results was that no pH detector was available to me to give me precise and quick results of the pH levels used. Instead I had to check by colour matching a pH strip, with the colour that the pH indicator said each pH level colour should look like. This means that the pH could if it was 6 for example, a range from 5.5 - 6.5 could be the actual pH level. As long as my eyes matched the colour indicated with the colour on the container, was what I thought the pH to be.
A third limitation to my experiment how many times I repeated my experiment. If I repeated it 3, or even 4 or 5 times, this would bring back results that would show reliability because of a greater amount of results for a mean to be calculated. Also to prove that my hypothesis worked even greater would be to use a wider range of pH levels. I would test above pH 9, at pH 10,11, 13 and 14. I would only be able to test this if I found a buffer that did not react with iodine, like pH 12 did.
I would add these changes if this investigation was to be carried out by me again, providing the resources were available. Further research could be done on how pH affects other enzymes besides alpha amylase. Because my experiment was on enzymes, this leaves room for further investigation to be implemented. Other variable like temperature, enzyme concentration, substrate concentration, the affect of competitive and non-competitive inhibitors are all possibilities on the affect of enzymes.
Concluding my investigating, my results support my hypothesis. At a pH level either too high or too low level would denature the enzyme. This was received at pH 2. The pH has an affect on enzymes as the three-dimensional shape that enzymes molecular structure is based on, is partly the result of hydrogen bonding. PH, which is a measure of hydrogen ion concentration, will affect the enzyme by changing the concentration of hydrogen in that enzyme. Any change in pH can effectively denature enzymes.
My anomalous results were identified and a justification for the reason behind it was explained. I see my results to be reliable due to the matching results of the repeated experiment. I explained the limitations to my experiment, and suggested improvements and alterations. This included the use of a pH buffer, and repeating the experiment further. Suggestions for further extended research were also given.
Bibiography
Student express. Properties on enzymes [online]
http://www.studentxpress.ie/educ/biology/bio4.html
Worthington biochemical organisation. The effect of pH on enzyme activity [online]
http://www.worthington-biochem.com/introBiochem/effectspH.html
Brooklyn university. The effect of pH on enzyme activity [online]
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm
Crocodile clips revision. Information on Catalysts [online]
http://www.crocodile-clips.com/absorb/AC4/sample/LR1505.html
Glenn & Susan Toole (1999) Biology for advanced level - fourth edition. Stanley Thornes
