Fair test:
I used the same amount of components each time I did the experiment and made sure that the test-tube aligned the same because the thickness of the glass can affect the light transmission. I will check the pH of the buffer I use to make sure that I am using the same each time. Although it is labelled, it can get contaminated with use.
Diagram:
Results:
Anomalous results are high lighted.
Analysis:
On average, the pH increased slowly, from pH 2 to 4, rising only 5% overall (denatured enzyme). At pH 7 however, it takes a giant leap to 87% (not including anomalous result), this is a difference of 79% to the sample tested at pH 4. The transmission stays high for pH 8 and takes a plunge there after, the difference of light transmission for pH 8 and pH 9 is 46% (R-type affected, slower rate of reaction).
Amylase is found in the mouth and the small intestine, both of which have a neutral to slightly alkaline pH (in the small intestine, this is for neutralizing stomach acids) and therefore has an optimum pH of around 7/8. Extreme pHs cause irreversible denaturing of the active site whist mildly incompatible pHs slow down reaction but is reversible once the charges have been balanced out.
Amylase is an enzyme, therefore it is a protein, and like all proteins, it consists of long strands of amino-acids, in an enzyme, these are made into a 3D shape by weak intermolecular bonds. The active site of an enzyme is specific to certain substrates and the enzyme is specific to certain pHs.
The way pH can reduce the rate of reaction is by changing the charge around the active site. Acids contain H+ ions, they are strong oxidizing agents whilst alkalis contain OH- ions, they are reducing agents. This can affect the ionic bonds that are holding the enzyme in shape and may also affect the R groups in the active site which form temporary bonds with the substrate.
There will be a pH, special to each enzyme, at which the net charge on the molecule is zero. This is called the isoelectric point (pI). The charge and charge distribution on the substrate and product will also be affected by pH changes in a similar manner to the effect on enzymes. Increasing hydrogen ion concentration will, additionally, increase the successful competition of hydrogen ions for any metal cationic binding sites on the enzyme, reducing the bound metal cation concentration. Decreasing hydrogen ion concentration, on the other hand, leads to increasing hydroxyl ion concentration which compete against the enzymes' ligands for divalent and trivalent cations causing their conversion to hydroxides and, at high hydroxyl concentrations, their complete removal from the enzyme. The temperature also has a marked effect on ionisations, the extent of which depends on the heats of ionisation of the particular groups concerned
In alkaline solution (pH > 8), there may be partial destruction of cystine residues due to base catalysed β-elimination reactions whereas, in acid solutions (pH < 4), hydrolysis of the labile peptide bonds, sometimes found next to aspartic acid residues, may occur.
The ionisation of the carboxylic acids involves the separation of the released groups of opposite charge.
The ionic strength of the solution is an important parameter affecting enzyme activity. This is especially noticeable where catalysis depends on the movement of charged molecules relative to each other. Thus both the binding of charged substrates to enzymes and the movement of charged groups within the catalytic 'active' site will be influenced by the ionic composition of the medium.
Evaluation:
My experiment did show what I was trying to find out, that amylase has an optimum pH of around 7/8. It is adapted to be positioned in the slightly alkaline conditions of the mouth. The results matched my predictions and the information I had found out on the topic.
It could have been a fairer test if the temperatures were the same for every experiment because in my experiments, I didn't use a water bath and the temperature of the room was slightly different every time I did a repeat of the experiment. I could have used a water bath at 37C so that the enzyme was working at its optimum temperature so that temperature differences wouldn't be an issue.
Also, I could have measured out the amounts of each component using a burette instead of a syringe for more accuracy.
In the future, if I were to do the experiment again, I would test all the buffers for accuracy before I used them, use a water bath to keep a constant temperature and a burette to measure the volumes of the solutions added. I would add the amounts of components differently so that I can use a conversion graph to work out the amount of starch in the solution.
My procedure would go like this:
- Mix three drops of standard iodine solution with 9ml of distilled water. Swirl to mix well.
- Insert tube into the colorimeter and switch on the meter. Rotate sample tube to get greatest possible deflection of the meter needle. Put a mark on the side of the tube with a china-graph pencil to indicate the position in the colorimeter and to prevent the mark washing off. Put the tube in the same position from now on.
- Adjust the dial to give a full scale deflection with the sample tube still in the meter. The meter should read either 100% light transmission or zero optical density.
- Empty out test-tube and wash well.
- Place 5ml of concentrated starch and 2ml of amylase into a water bath at 37 degrees Celsius and leave for 10 minutes.
- Add 2ml of buffer, the starch and then 3 drops of iodine to the test tube. (Test buffer for pH before hand)
- Mix well and add the enzyme, mix well and take the reading on the colorimeter after 3 minutes.
- Repeat this with all the other buffers.
- Remember to wash test tube thoroughly in-between each experiment.
I think that my results were quite accurate; I was able to get clear, unambiguous readings from the colorimeter. It is also reliable because I had repeated the tests five times and I have gotten quite similar results.
I did get one anomalous result, but then I realised it was because the buffer had been contaminated, I tested it with universal indicator solution and found that instead of being pH 7, it was slightly acidic.
In any future experiments, I would also use a wider range of pHs to get a more accurate and reliable graph. However, in this experiment, the buffers were unavailable at the time so I could not use them.
