Investigation on the effect of pH on enzymes

Design Experiment.
How does pH of potatoes affect the breakdown of starch by the enzyme Salivary Amylase? Enzymes are proteins that are found inside our bodies and their roles are to break down large molecules into smaller soluble ones. Enzymes are found in different parts of our bodies and each of them breaks down a specific type of substrate at specific conditions. These conditions include pH and temperature. Our internal body temperature is around 37°C and this is the optimum temperature for our enzymes to functions, meaning they work their best at this temperature. This means that the enzymes cannot function properly if the temperatures they are exposed too is too high or too low. High temperatures actually denature enzymes, where they cannot function at all as their active site (area that binds to the substrate) changes shape. pH is something that differs in our body, for example: the pH in our stomach is very acidic due to gastric juices whereas the pH in our mouths is almost neutral. This means that enzymes found in a stomach are adapted to work best at very low pH and therefore cannot work in a pH that is higher, example: pepsin, produced in the pyloric glands in the stomach walls. Salivary amylase is produced by the salivary glands found in our mouths, they work best at almost neutral pH, so they cannot work when the pH becomes too high or too low. Potatoes (Solanum tuberosum) are tubers and therefore are the starch stores for plants, meaning they contain a lot of starch.

When iodine solution reacts with a starch rich substance it goes very dark blue/ black. The enzyme salivary amylase breaks down starch into maltose. As the salivary amylase breaks starch down the test of starch using iodine will produce colors that are lighter than dark blue/black, as there is less starch present. This investigation aims to determine how different pHs affects the action of salivary amylase on starch by analyzing how it affects the color change of the iodine solution on thin strips of potatoes that were soaked in different pHs, ranging from acid, neutral and alkali environments. This investigation will also confirm which pH salivary amylase works best and worse. Hypothesis:

As the pH becomes too low or too high (too far from neutral [7], which is the optimum pH for salivary amylase) the enzyme: salivary amylase will have a lower rate of reaction meaning the stain of iodine on the potato piece will become darker as less amylase reacts with the starch in the potato. This is because salivary amylase works best at an almost neutral environment like that found in our mouth.

Dependent variable:
The color intensity of the iodine solution on starch present in potato, using a key to identify the color intensity. This key will provide data that is quantitative rather than only qualitative. The key will consist of 4 different color intensities which correspond to a numeric value: Dark Blue/Black

This scale shows that the smaller the value the smaller the amount of starch present. This variable will be controlled by ensuring that each potato piece has the same mass, and roughly the same thickness, as this can affect the amount of starch in the sample, and therefore make results varied. This variable does not include uncertainties since no equipment will be used to generate the results. This means that this data is subjective and therefore very prone for errors and inaccurate results, as there can be different interpretations as to which value a color corresponds to. This also means that this experiment needs to be repeated many times to ensure a large range of data.

Independent variable:
The pH in which potatoes will be exposed (pH) (± 0.05).
The potatoes will be exposed to different pHs, ranging from 2,4,6,8 and 10. This variable will be controlled by using a pH probe to ensure that the pH of the solutions are always the ones wanted, so that they do not vary when repeating the experiment for averages. The pH must be kept the same since pH is a limiting factor for enzymes. If pH are not exact the results will be tainted and a conclusion cannot be drawn. All the pH will be done in isotonic solutions so that the osmolarity of the potatoes does not change, as this could lead to misleading results. It will be used isotonic solution of Hydrochloric acid for lower pH, isotonic water solution for neutral pH, and an isotonic Sodium Hydroxide solution for the alkali solution.

Controlled variables:
Temperature (°C )(±0.05) of the potato and of the amylase solution. This variable is going to be controlled using the potato and amylase solution at room temperature (37°C), because this is an accurate way, using a bulb thermometer, to maintain their temperatures and it is a suitable temperature for amylase to work in (although not optimum). This variable must be controlled as temperature like pH can also effect the action of amylase. If the temperature is too low the enzyme cannot function well, and if it is too high it gets denatured then cannot function at all. pH (pH) (± 0.05) of the salivary amylase solutions .

This variable will be controlled using a pH probe. The pH of the amylase solutions must always remain constant to ensure a fair test and will be kept at neutral pH (7) since this is the pH of our mouth and the place where salivary amylase works. Size (length and width) of potato piece used to test (mm)(±0.05). This variable is going to be controlled by using a scalpel to cut out a 1 cm piece of potato and ensuring that its width throughout is 3 mm. If the potato samples differ from each other in thickness the results will be affected as the test will not be a fair one, because it can affect the color of the potato, for example in a thin piece of potato a dark blue color may appear lighter than a dark blue color in a thicker piece, as there is more of the color. For the results to be the most accurate possible the potato slices must be very similar so that the amount of starch initially present is almost the same, this way the effectiveness of the amylase can be compared equally, this means that the pieces must always come from the same part of the potato, such as the middle part. The mass of the potato (g) (± 0.005).

This variable will be controlled using a scale, and it is to check that all the potato pieces have the same mass so that the test can be fair. The mass should be around 30 g for each piece. Concentration (mol/dm3) of iodine used to stain potato.

This variable is going to be controlled using a new iodine bottle with the concentration prepared by a company, which will be 1%. If different concentrations of iodine are added the colors will differ from each other and will no longer be useful as their colors are affected by the amount of iodine present. Since all the samples will be compared to each other it is critical that the same concentration of iodine is used each time. Volume of iodine used to stain potato (drops).

Since the potato pieces must only be stained using 3 drops of iodine this variable will be controlled using a graduated pipette. Amount of time (sec) (± 0.00005).  Each potato must have the same time (5 minutes) submerged in different pHs solutions to ensure a fair test. This is important because if one sample of potato is allowed to soak in a solution for more time it may become more acidic or more alkali that it should and therefore taint results. Each potato must also be during the same time (6 minutes) in amylase solution, so the time of enzymatic reaction is the same . Concentration (mol/dm3) and volume (ml) (± 0.005) of amylase solution. The concentration of the solution will be 1% and the volume will be 3ml. The enzymes will be acquired from a company and the concentration will be stated in the new bottle. The volume will be controlled using a graduated pipette.

Apparatus and materials:
pH probe (% transmission) (± 0.05)(ensure same pH for each test) 10 potatoes (for repeating the experiment) 1 scalpel Scale(g) (± 0.005 grams) prepared isotonic solutions with pHs ranging from pH 2,4,6,8 and 10 250 ml of iodine at 1% concentration key for color intensity ( dark blue/black =8, light blue/black=6, brown=4, light brown/yellow = 2) stopwatch (+/- 0.00005 seconds) white tile (to have a good contrast between color of potato slice and white tile) 6 pipettes ( one for the iodine solution, then one for each isotonic solution) Tweezers 250 ml of Salivary Amylase at 1% concentration 10 petri dishes Ruler (cm/mm)(±0.05)
Thermometer (◦C)(±0.05)

Methodology:
1. Devise a key that can be used to evaluate the color intensity of the iodine solution stain on starch in potatoes to analyze the effect pH has in amylase. 2. Cut a potato slice that has a length of 1 cm, and width of 2 mm. Record its mass which should be around 30 grams (all the remaining potato slices must be similar to the original piece in terms of mass, width and length). 3. Leave the isotonic solutions, the potatoes and the amylase solution in the room, and only start the experiment once these have all reached room temperature (use a thermometer to check they are all the same temperature). 4. Once it has reached room temperature add 10 ml of the isotonic solution of pH 2 to a petri dish and then soak the potato slice in it for 5 minutes (the potato piece must be completely submerged). Add a pH probe is the solution to monitor the pH.

5. After 5 minutes soaking remove the potato and place it on a clean petri dish. 6. Add 3 ml of amylase solution to the potato in the new petri dish and let it react or 6 minutes. 7. After the 6 minutes, remove the potato slice using the tweezers and place it on a white tile. 8. Now add 3 drops of iodine solution to the potato slice, and using the key evaluate what the color intensity is, write it down on the results table. 9. Repeat steps 2-7 for each pH, and record all the values on the results table, a full set of data should be acquired. 10. Now repeat the experiment 20 times for each pH in order to establish enough data so that an average value can be taken and so a more accurate graph can be produced. And so standard deviation can be established to produce error bars on the average graph of the results. 11. Present the results in a bar graph so values can be easily compared and so the conclusion can be reached. Equipment setup: