The aim of this investigation is to find out how different pH levels affect enzyme activity.

Amylase PH Experiment

Aim

Introduction

Enzymes are globular proteins made up of amino acids strings that are twisted into elaborate shapes. They act as biological catalysts and are soluble. The shape of the enzyme is vital, as the substrate/s must fit exactly into the active site (a cavity) of the enzyme, rather like a lock and key. Once it is inside it forms an enzyme-substrate complex. Enzymes are either catabolic or anabolic. Catabolic enzymes break the substrate down into 2 or more products while an anabolic enzymes bond 2 or more substrates together to form one product. There is also an enzyme induced-fit theory which states that the enzyme changes shape to accommodate the substrates shape and then goes back to its original shape after the reaction is over. Enzymes never get used up so they keep on being re-used until the reaction is over or they get denatured. Enzymes are also specific; they only work on one type of enzyme, hence the specific shape of the active site.

Enzymes are affected by changes in temperature and pH. The optimum temperature of enzymes is at 37C and they become denatured (because the bonds holding the amino acids strands begin to break which results in the change of the active site) at high temperature. At lower temperatures enzymes still work although not best.

Enzymes are very useful in our day-to-day lives. They help digest food and are produced in our mouth, pancreas and stomach. They break down larger molecules into smaller ones so they can be absorbed into out blood through the small intestine wall. Amylase breaks down starch molecules into simple sugars.

Enzymes work by lowering the activation energy, which is the amount of energy required to make or break a bond. This is why they work much faster than reactions without enzymes and are time effective.

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pH is the measure of hydrogen ion concentration in a solution, which gives us the solutions acidity. It is measured on a value of 1-14 with 7 being neutral. Water has a pH of 7 while hydrochloric acid is an example of a strong acid, while sodium carbonate is an example of a strong alkali. The more hydrogen ions there are in a solution the more acidic it becomes and its pH value lowers. Extremes of pH will cause bonds between the secondary and tertiary structures to break therefore denaturing the enzyme. Smaller pH changes result in the R group in the amino acids to become ionised therefore changing its shape and in turn resulting in a change in the shape of the active site. Most enzymes work best at neutral pH but some work best at slightly acidic or slightly alkali conditions.

Enzyme concentration also affects the rate of reaction of an enzyme. Solutions with a higher enzyme concentration work faster because they have more enzymes available to be used up and therefore the reaction happens quicker. Substrate concentration has an effect on the rate of reaction when the amounts of substrate far out number the amount of enzymes. Because there are only so much enzymes more substrate will have to wait till an enzyme becomes free, so the only way to make that reaction happen faster will be to increase the enzyme concentration, although because enzymes work very fast it is unlikely this situation will arise.

Hypothesis

I believe that as the pH value of the solution progresses to 7 the rate of reaction will increase and the enzymes will be working at optimum speed at 7. pH is the measure of hydrogen ion concentration in a solution, which gives us the solutions acidity. The more hydrogen ions there are in a solution the more acidic it becomes and its pH value lowers. As the pH value of the solution shifts away from 7 the rate of reaction will start to decrease. This is due to the fact that amylase has an optimum pH of 7. Changes in pH cause ionisation on the R groups of amino acids, which changes the shape of the enzyme and in turn the shape of the active site, and so the substrate/s will not be able to fit into the active site.

I will use the following apparatus:

I will keep the following controlled variables constant because they will affect the rate of reaction of the enzyme is not kept in check:

Temperature of solution

Substrate concentration

Enzyme concentration

The temperature will be kept at a constant 37C because that is optimum temperature for enzymes. In order to keep it at a constant 37C I will keep the test tubes containing the solution in warm water, which will be constantly checked, and if the temperature changes than more warm water will be added to make sure it stays at 37C. I will use a thermometer to check on the temperature.

The substrate concentration will be kept the same, as I will be using the same starch solution and also the same amount with all the different pH values. It will be at 50% because half of the starch solution will be water. This will ensure that the enzymes have enough substrate to work with and so they will be working at maximum output.

The enzyme amylase concentration will also be kept the same at 0.5% because it seems a reasonable concentration value and also if I increase it more the reaction will happen very quick and I don’t want it to happen instantaneously as I want results that can be clearly seen on a graph from which patterns and conclusions can me made.

For the pH values of the solution I will use pH values 5,6,7,8,9 because they are a good balance of values and will make my results more accurate. I will also repeat each experiment thrice therefore giving my results more credibility and reliability. I will calculate the average of the three values and plot that on the graph, unless of course a result is extremely anomalous, then I would see if there was a reason for this and whether it should be included in the average value.

My apparatuses are simple and fairly precise but the 10cm syringe has quite a large range for error (±2mm) so I will have to be careful with my measurements to make sure there precise, because then there will be a greater chance of the results being more accurate.

My number one concern will be safety and the safety rules must be followed at all times if the experiment is a success. For safety reasons I will wear goggles and any long hair will be tied back and also a lab coat will be worn. I will also be careful with the HCl, as this is acidic and may corrode the skin if in contact, so extra measures should be taken to avoid spillage and handling of glass. Normal lab rules will apply like no running, chewing etc.

I will write my results on a table and work out the rate of reaction using the results as well as drawing all the relevant graphs

Method

I will start by measuring the pH of the solution by titration, which will neutralise the acid by a certain quantity of base of known concentration. This will all take place n the presence of an indicator whose colour depends on the pH. I will then be able to tell what ph the solution is at and either add acid/alkali to get it to the pH level that I need. I will label the 5 test tubes as pH 5, pH 6, pH 7, pH 8 and finally pH 9. I will get the dimple trays and empty 2 drops of iodine solution into each cavity. Thereafter I will get a 10cm syringe and fill it up with starch solution and empty 9cm of it into a test tube. I will repeat this for the other 4 test tubes and place the test tubes in a beaker containing water at 37C using a thermometer to check the temperature and adding hot/cold water if need be. Then I will get the 1cm syringe and fill it up with the relative pH solution and empty it in its specific test tube, so the pH 5 solution will be in the pH 5 test tube. I will give the test tube a shake and simultaneously start the stopwatch. I will then use the 1cm test tube to fill up 0.1mm of the solution and when the time reaches 30s I will empty the contents of the syringe into the cavity. If the colour is black I will again fill 0.1mm of the solution and after another 30s I will repeat emptying the 0.1mm into the cavity to see if it turns mid-brown. Mid-brown will be my end point as this will notify me that the starch in the solution has been broken into sugar molecules hence the iodine solution not being blue/black anymore. I will write the time taken on a table and repeat this experiment 2 more time for pH 5. Thereafter I will start from the beginning with pH 6 and follow the instructions above, and likewise do the experiments for 7,8 and 9 three times each.

I will keep an eye on the controlled variables to make sure they are constant in all experiments in order for it to be a more fair and reliable test.