The effect of temperature on the action of the enzyme trypsin

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Biology Coursework – Enzymes

The effect of temperature on the action of the enzyme trypsin

Aim

To investigate the activity of trypsin under the change of temperature.

Introduction

Enzymes are catalysts in the body that speed up the breakdown of food, and are essential in the digestive system. Although food can be broken down by molecules colliding with it, the process is speeded up greatly by enzymes, a type of catalyst. We know from previous knowledge that enzymes are proteins, and they require the presence of other compounds, or co factors, before their catalytic ability can be exerted. Enzymes can be used more that once and are replaced only after a period of time when they are denatured. They are all specific to certain foods, but some are more specific than others. The lock and key theory of enzymes is that each enzyme has a specific ‘key’ shape, which will only fit into one sort of ‘lock’, or substrate. This is illustrated in the below diagram.

We also know they are denatured by high temperatures, certain salts, solvents and other reagents, where they lose their ‘lock and key’ shape, making them useless. Enzymes work by attaching themselves to a bond in the substance and breaking the bond between them. It is because of these reasons we chose to examine enzymes in this experiment. The enzyme we are using is trypsin, which breaks down amino acids in the body.

Hypothesis

I hypothesise that as the temperature is increased, the rate of reaction will increase. However, when higher temperatures are reached, enzyme reaction rate will drop rapidly as the enzymes are denatured. I have drawn out a hypothesis graph, which I believe will be what the final graph will look like approximately. I have also labelled a number of stages, and explained what will happen to them, as known from past experience and research.

We know that even without enzymes in the solution, the rate of reaction would still increase, due to the kinetic theory. The higher the temperature, the more heat energy. Heat energy is converted into kinetic energy, making the water molecules move around more quickly, hence colliding more often with the substrate, helping break it down. We also know that if enzymes are present, when the temperature is higher, the substrate will move more quickly into the active site. At stage 1 on the graph, the rate of reaction increases at a similar rate to the temperature, that is, the increase is roughly proportional. At stage 2, the enzyme activity is at its peak, I believe around 40 degrees. At stage 3, the enzyme is becoming denatured from the high temperatures, and is losing its unique shape, which allows it to catalyse substances. At stage 4, the high temperatures have completely denatured the enzyme, making it ineffective as a catalyst. 20o

Variables

There are a number of different types of variable in this experiment. The independent variable in this test is the temperature. We ensured that this stayed exactly as we wanted it by checking the temperature before inserting the photographic film. This is the only variable we should be changing and have direct control of. The dependant variable is the result we want, that is, the rate of reaction. If we perform the experiment correctly, the only thing that should be affecting the dependant variable is the independent variable, the temperature. All other variables, such as pH and the size of the photographic film, should be kept constant to ensure a fair test. If we change the pH, the enzyme’s bonding will change and cause it to lose its active site. This would make the test unfair, so we added buffer to the solution to keep the pH constant. If the photographic film size was not constant, it would take longer for the enzymes to break down the gelatine on some of the film, while the experiments with smaller pieces of photographic film would be broken down faster. To ensure all conditions are identical apart from temperature in each test, we kept the size of the photographic film to exactly 4mm squared. We also ensured that all tubes were given 10 minutes to acclimatise to the appropriate temperature. This is discussed later in detail. The temperature, the main variable we will be controlling, we have decided to test the trypsin at 0 degrees, 20 degrees, 40 degrees, 60 degrees and 80 degrees. We decided to choose this range, as it should provide a large spectrum of results, and are at the same time not too far apart in temperature, so we can hypothesise what results between them will be once we have our set of results. We made the temperatures precise by using water baths set to exactly the correct temperature, or an icebox at exactly 0 degrees. We also used a thermometer to ensure the temperatures were correct just before starting the experiment.

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Procedure for Preliminary Test

Apparatus:

  • Test tube rack
  • 10 Test Tubes
  • Ice for 0 degrees experiment
  • Thermometer
  • Water baths at 40, 60 and 80 degrees.
  • Trypsin solution
  • 10 pieces of photographic film
  • 10 splints with cut ends
  • Stopwatches

Method

Before beginning, we ensured the area was safe by wearing safety goggles and clearing the nearby area of books or obstacles. We placed 10 test tubes in the test tube rack. 5 were control tubes, so we added exactly 3ml of water to each of the 5. We then added 3ml of ...

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