Effects of environmental temperature and pH on porcine pancreatic amylase activity

Porcine Pancreatic Amylase

Effects of Temperature and pH on Porcine Pancreatic Amylase

Enzymes are incredibly important chemical substances that allow for and many time catalyze certain reactions within the body that drive the basic processes of life. Almost every molecular process that occurs in a living body is dependent in some way, whether directly or indirectly, on the function of certain enzymes, from the digestion of food to the complex process of cellular reproduction, enzymes play a vital role in the continuation of life and its processes (Worthington 2009). The way they do this is by binding to certain other molecules being used and/or altered in the reaction; the substance that directly reacts with an enzyme is called the substrate (Nuffield 2009). The substrate binds to the enzyme on the enzyme's active site, and this generally allows whatever change to the molecule needs to occur at that particular point in a chemical process to occur (Nuffield 2009). If the conditions aren't right, however, there could be a serious interruption to the functioning of the enzyme, which is one of the reasons that homeostasis is so important -- maintain proper balance in the body allows enzymes to function.

Though most enzymes are fairly hearty and can operate in a wide range of environments, they perform much better when their optimal conditions are met (Worthington 2009). Two especially important factors are the temperature of the environment, and the pH or acidity here the reaction is taking place. Again, most enzymes can operate in a wide range of temperatures and pH levels, but there are certain optimum ranges that can be quite narrow -- especially in the case of pH -- in which the enzymes operate most efficiently (Worthington 2009). These narrow ranges tend to correspond to the levels found in the certain areas/organs of the body where a given enzyme operates, making life more efficient (Nuffield 2009).

Amylase is one of the over 700 enzymes at work in the human body, and also appears in many other species as its purpose is widely needed -- amylase breaks down otherwise indigestible starch into sugars, which can then be further broken down into the glucose that animals can use for energy (Allsands 2007). In the following two part experiment, I will attempt to determine the optimum pH levels and temperature in which amylase operates -- that is, what conditions provide for the most efficient conversion of the substrate (starch) into the product of the reaction (sugars) by the enzyme (amylase). My first hypothesis is that temperature will not have a major effect on the reaction rate except at extremes (i.e. near boiling and near freezing temperatures). Secondly, since I will be using porcine pancreatic amylase, I hypothesize that the optimum pH level will be that found in the average porcine pancreas, which is slightly more acidic than neutral (Nuffield 2009). Finally, I hypothesize that differences in pH will result in far greater differences in reaction times than will temperature.

Methods

In order to perform this experiment, several solution will be needed. First, a solution containing 1% amylase, which is a high enough concentration to operate but a very low safety concern (Nuffield 2009). In addition, a starch solution must be made for the enzyme to act upon, as well as a series acid solution or buffer to alter pH levels, and finally an iodine potassium iodide solution to test the results (Nuffield 2009). This last solution is orange/amber in color, but turns a dark blue in the presence of starch, meaning it can be used to measure when the reaction is completed and no starch is present in a sample (Allsands 2007).

To perform the experiment, iodine solution was placed in a series of tiles containing individual wells. For the temperature portion of the investigation (conducted first), eight test tubes were placed in four temperature controlled water baths ranging from water and crushed ice (2( C) to near boiling (98(). The other baths were kept at room temperature (23( C) and an intermediary between this and boiling (75( C). Test tubes were left in the baths for ten minutes, until their temperatures at equalized. Starting with the coldest bath, the amylase mixture was poured into the starch solution and briefly stirred. Leaving the test tube in the bath, a single drop was removed every ten seconds using a plastic pipette and placed into a well containing the iodine solution. When the color remained orange, the reaction had been completed, and the time (i.e. length of reaction) was noted. This same process was repeated with the other three sets of test tubes at the other temperatures, washing and replacing the iodine trays when needed.

To test for pH efficiency, different concentrations of the buffer solution were created at pH 3,5, 7, and 9. These were added one at a time to four separate test tubes of starch solution, and then the amylase solution was introduced. Again, drops of the combined solutions were transferred to wells on the iodine tray, and when the iodine solution remained orange the reaction was considered complete and the time noted.

Results

The results of the temperature portion of the experiment appeared fairly straightforward. At near freezing temperatures, the reaction took nearly eight minutes to complete. This time was cut in half at room temperature, and was diminished significantly again at 75( C. At 98( C, (near boiling), however, the reaction never took place. After ten minutes without change in the iodine solution, it was assumed that the amylase had been effectively destroyed by the high temperature and that no reaction was taking place.

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Temperature

In the pH experiment, the results were lightly more complex; rather than a simple increase in the efficiency of the reaction, a noticeable dip in the reaction time occurred in the neutral pH sample (pH 7), with effectiveness limited only somewhat by the higher pH but more noticeably reduced in the lower pH solution.