Determining the Optimum pH for Trypsin

Introduction
Trypsin is a kind of protease. This enzyme is present in the small intestine and can break down protein into amino acid. Different enzymes may have different optimum pH levels. At the optimum pH, the enzymes work best. The activity is the highest. In lower pH or higher pH, the excessive hydrogen or hydroxide ions may break the ionic bonds. This changes the shape of the enzyme. The shape of the active site also changes. This lowers the catalytic activity. Photographic film has a protein called gelatin that coats its surface. If it is removed, a whitish stain will appear.

Principles
In this experiment, we place a photographic film strip into a trypsin solution. The protease will digest the gelatin coating and make it whitish. Different buffer solutions will be added to the solutions as well, to change the surrounding pH. The degree of digestion of the gelatin reflects the activity of the enzyme. Therefore, the optimum pH can be estimated.

Procedure

1. Set up the water bath and adjust the temperature to 37°C.
2. Pipette 1 mL buffer solution of pH 1.0 into a test tube.
3. Add a strip of photographic film into the solution and put it into the water bath for 5 minutes.
4. Pipette 1 mL of trypsin solution into another test tube. Put it into the water bath for 5 minutes.
5. Pour the 1 mL of trypsin solution into a test tube with film.
6. Record the time it takes for the complete disappearance of gelatin on the film.
7. Repeat the experiment with buffer solutions of pH around 2.0, 4.0, 6.0, 8.0, and 10.0.

Precautions

1. The temperature must be kept constant, since temperature is a factor affecting the enzymatic activity.
2. The gelatin coating is easily scratched and damaged, and should be handled with care.
3. The enzyme and the film have to be put into the water bath for 5 minutes before mixing. This allows both substrates and enzymes to reach the optimum temperature before mixing.
4. The tubes must be mixed from time to time.
5. If the gelatin coat remains for over 1 hour, we can assume the time to reach the optimum pH to be infinity.