Lab Report: Effect of Temperature Changes in Trypsin Proteolytic Action on Proteins

• Introduction

Proteins are remarkably heat-sensitive, and their functions can be enhanced or diminished by changes in temperature. At extreme temperatures, many proteins become denatured and lose their function or together. Temperature changes affect the functioning of various proteins, including enzymes. Makhatadze (2007) reports thermodynamic changes regarding a protein’s heat capacity, enthalpy, entropy, and Gibb’s energy change. All these properties share a linear relationship and contribute to the configuration of a protein, determined primarily by the degree of protein unfolding.

Essentially, all proteins undergo conformational changes that influence the rate of activity of enzymes when temperatures change. High temperatures stimulate conformational changes, allowing enzymes to react with substrates and cycle through active and inactive states. Because different proteins have different thermodynamic properties, each enzyme has its spectrum of activity and inactivity. The trend is generally for proteins to increase in action at a steady rate as temperatures rise and for the trend to reverse as temperatures fall.

This experiment assesses the effect of heat and temperature changes on the action of trypsin on proteins. Trypsin is a protease enzyme that in humans is released in the gastrointestinal tract to digest proteins into smaller peptides and amino acids. Trypsin was exposed to different temperatures in this experiment, and reactions were recorded as color changes. A color change to yellow confirmed the protease activity of the trypsin following the hydrolysis of benzoylarginine p-nitroanilide (BAPNA). BAPNA served as an indicator for the proteolytic action of trypsin. Enzymatic action was evaluated at normal body temperature and freezing temperatures after exposing the enzyme to boiling temperatures. One of the hypotheses was that the reaction would be faster in the presence of enzymes. The reaction would also be much slower at body temperature and more rapid at boiling temperatures. However, there would be no reaction at freezing temperatures.

Materials and Methods

To conduct this experiment, several items must be made available. A rack of test tubes with at least 5 test tubes is required. The test tubes will hold the substrate, enzyme, and indicator. A hot plate is also needed as a heat source with which to boil the trypsin solution and maintain the water bath at 37 degrees Celsius. A large beaker for boiling shall hold this water bath, and the test tubes will be inserted into the water bath. Disposable droppers will be necessary for adding water, BAPNA, and trypsin to the test tubes. This experiment’s reagents include trypsin, BAPNA, and distilled water. A marker is also necessary for labeling the test tubes.

First, the test tubes are labeled 1 to 5 and placed on the test tube rack. Or the first two test tubes, 2 milliliters of water are added to each test tube. In the first test tube, 2 milliliters of trypsin are added, and the tube is set aside. To the second test tube, 2 milliliters of BAPNA are added, but no trypsin is added. These first two test tubes serve as controls for the experiment. No color changes are expected in either of them. For the third, fourth, and fifth test tubes, 2 milliliters of BAPNA solution are mixed with 2 milliliters of trypsin. When a reaction between trypsin and BAPNA occurs. There should be a change in color to yellow. This color change should be recorded. The third tube contains trypsin that has been boiled for 4 minutes. This tube is incubated at 37 degrees Celsius for 1 hour. The fourth tube contains a simple trypsin solution that has not been heated. This tube is also incubated at 37 degrees Celsius for an hour. The fifth tube is also incubated at 0 degrees Celsius for one hour. Color changes for each tube are recorded.

Results

All the control tubes underwent no color change, while all the test tubes underwent a color change. The solution remained clear in tubes 1 and 2. It, however, changed to yellow in tubes 3, 4, and 5. After boiling trypsin for 4 minutes, the solution turned yellow after incubating it at 37 degrees Celsius for one hour. The solution also turned yellow when the solution was incubated. Even at freezing temperatures of 0 degrees Celsius, the solution still turned yellow. The results of this experiment are presented in table 1 below.   Trypsin-BAPNA Rection At Different Temperatures Milliliters Incubation Tube Water BAPNA solution Trypsin Solution Direction Color 1 2 0 2 Incubate at 37 degrees for 1 hour. No color change 2 2 2 0 Incubate at 37 degrees for 1 hour No color change 3 0 2 2 Boil trypsin for 4 minutes incubate at 37 degrees for 1 hour Yellow 4 0 2 2 Incubate at 37 degrees for 1 hour Yellow 5 0 2 2 Incubate at 0 degrees for 1 hour Yellow

Table 2. Table Showing the Color Change of BAPNA’s Reaction with Trypsin at Different Temperatures  

Discussion

This experiment is meant to demonstrate the proteolytic activity of an enzyme through its action on BAPNA. When BAPNA undergoes hydrolysis, the solution changes from colorless to yellow, indicating that the enzyme is active. The first two test tubes underwent no color change as proteolysis did not occur. In the first, there was no BAPNA, hence no substrate for the enzyme. No enzyme was present in the second test tube, so proteolysis also did not occur. There was a color change in all the other test tubes. In tube 3, the expectation was that boiling trypsin at 100 degrees Celsius for four minutes would denature the enzyme and prevent it from reacting with BAPNA. No color change was expected for this reaction. However, the solution turned yellow, demonstrating that trypsin is heat-resistant and preserves its proteolytic action even at high temperatures. Tube 4 also turned yellow. The tube was incubated at 37 degrees Celsius, close to the normal body temperature of humans. The reaction occurred rapidly, demonstrating that trypsin is active at physiological temperatures. Test tube five also changed color despite being incubated at zero degrees. This finding indicates that trypsin retains its proteolytic activity even at low temperatures.

The experiment did not support the hypothesis that had been formed at the beginning of the experiment. Since the rate of color change and the degree of color change were not recorded for this experiment, it is impossible to determine the impact of temperature change on the activity rate or trypsin. However, the experiment showed that trypsin’s activity is preserved even after boiling and freezing. It was not possible to demonstrate that activity is optimal at body temperatures. It was also impossible to demonstrate the increased enzymatic activity with temperature increments. These results are significant because most proteins are heat-sensitive, denatured at supraphysiological temperatures, and inactive at low temperatures. This is not the case with trypsin. The experiment shows this enzyme’s resilience and that it is active over a wide range of temperatures. Organisms inhabiting high-temperature and cold regions can harness this enzyme’s digestive powers regardless of environmental conditions.

Clinical Implications

Some individuals might suffer from disorders affecting the action of trypsin in their digestive systems. It is therefore essential that their pancreatic secretions are investigated for trypsin activity. These secretions can be tested with BPNA incubated at 37 degrees Celsius and looking for a color change to yellow.

• References

Makhatadze, G. I. (2007). Thermodynamic Properties of Proteins. Physical Properties of Polymers Handbook, 103–143. https://doi.org/10.1007/978-0-387-69002-5_8