Effects of Temperature and pH on Porcine Pancreatic Amylase

Introduction to Enzymes and Amylase

Enzymes are incredibly important chemical substances that catalyze reactions within the body, driving the basic processes of life. Almost every molecular process that occurs in a living body depends 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). They accomplish this by binding to specific molecules involved in a reaction; the substance that directly reacts with an enzyme is called the substrate (Nuffield 2009). The substrate binds to the enzyme at the enzyme's active site, which generally enables whatever molecular change is required at that point in the chemical process to occur (Nuffield 2009). If conditions are not right, however, a serious interruption to enzyme function can result — which is one of the reasons homeostasis is so important. Maintaining proper balance in the body allows enzymes to function correctly.

Though most enzymes are fairly robust and can operate across 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, at which the reaction takes place. Most enzymes can operate across a wide range of temperatures and pH levels, but there are certain optimum ranges — which can be quite narrow, especially in the case of pH — within which enzymes operate most efficiently (Worthington 2009). These narrow ranges tend to correspond to the conditions found in the specific areas or organs of the body where a given enzyme operates, making the organism's biological processes more efficient (Nuffield 2009).

Amylase is one of more than 700 enzymes at work in the human body, and it also appears in many other species because its function is widely needed: amylase breaks down otherwise indigestible starch into sugars, which can then be further broken down into glucose that animals use for energy (Allsands 2007).

Hypotheses

In the following two-part experiment, the goal is to determine the optimum pH levels and temperature at 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).

The first hypothesis is that temperature will not have a major effect on the reaction rate except at extremes — that is, near-boiling and near-freezing temperatures. Second, since porcine pancreatic amylase is used in this experiment, it is hypothesized that the optimum pH will correspond to that found in the average porcine pancreas, which is slightly more acidic than neutral (Nuffield 2009). Finally, it is hypothesized that differences in pH will produce far greater differences in reaction times than differences in temperature.

Methods

Several solutions were required to perform this experiment. First, a 1% amylase solution was prepared — a concentration high enough to drive the reaction while presenting minimal safety concern (Nuffield 2009). A starch solution was also prepared for the enzyme to act upon, along with a series of acid solutions or buffers to alter pH levels. Finally, an iodine-potassium iodide solution was used to test the results (Nuffield 2009). This solution is orange/amber in color but turns dark blue in the presence of starch, meaning it can be used to detect when the reaction is complete and no starch remains in the 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, eight test tubes were placed in four temperature-controlled water baths ranging from water and crushed ice (2°C) to near-boiling (98°C). The other two baths were kept at room temperature (23°C) and at an intermediary temperature between room temperature and boiling (75°C). Test tubes were left in the baths for ten minutes until their temperatures 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 was considered complete and the time — that is, the length of the reaction — was recorded. This same process was repeated with the other three sets of test tubes at the remaining temperatures, with the iodine trays washed and replaced as needed.

To test for pH efficiency, different concentrations of the buffer solution were prepared 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 was recorded.