To investigate the effect of Trypsin using a photographic film

Prediction: In this experiment there are two variables. Firstly, the pH. Changes in pH would alter or totally inhibit the enzyme from catalysing a reaction This change in the pH will affect the polar and non-polar intramolecular attractive and repulsive forces and alter the shape of the enzyme and the active site as well to the point where the substrate molecule could no longer fit, and the chemical change would be inhibited from taking place as efficiently or not at all.

Every enzyme has an optimum pH range outside of which the enzyme is inhibited. Some enzymes like many of the hydrolytic enzymes in the stomach such as Pepsin and Chymotrypsin effective operate at a very low acidic pH. Other enzymes like alpha amylase found in the saliva of the mouth operate most effectively at near neutrality. I think that trypsin will work best at an alkali pH like 8 or 9. I think this because trypsin works in your duodenum and this has an alkali pH.

Secondly the temperature. Every enzyme has a temperature range of optimum activity. Modification of the lock and key hypothesis assumes that the active site has a certain amount of elasticity whereby the active site can expand or contract in a limited way in order to accommodate the substrate molecule. The analogy is like a hand fitting into a glove. The glove adjusts in shape and size to fit various sized hands within a certain range. This tolerance would explain why bogus molecules of slightly different size compared to the true substrate molecule could still be accommodated by the elastic active site.

Small changes in temperature would distort the active site conformation but not so much that the active site could not still accommodate the substrate molecular size. PH changes which would also change the active site conformation but not so much that the active site could not flexibly accommodate the substrate molecule. The Induced Fit Model seems to explain why there is some flexibility in the edibility of the active site to accommodate other molecules and at limited temperature and pH ranges.

Outside that temperature range the enzyme is rendered inactive and is said to be totally inhibited. This occurs because as the temperature changes this supplies enough energy to break some of the intramolecular attractions between polar groups as well as the Hydrophobic forces between non-polar groups within the protein structure. When these forces are disturbed and changed, this causes a change in the secondary and tertiary levels of protein structure, and the active site is altered in its conformation beyond its ability to accommodate the substrate molecules it was intended to catalyse. Most enzymes (and there are hundreds within the human organism) within the human cells will shut down at a body temperature below a certain value, which varies according to each individual.

In this case I think that it will work best a 400 C as this is about body temperature and it is at about this temperature that it works in the Duodenum.

Preliminary experiment:

We are investigating the best pH at which Trypsin works.

Method:

A beaker was filled with water of about 400C. Each test tube was filled with 5cm3 of trypsin and 5cm3 of water with a buffer solution one pH 4 one pH 7 and the other pH 9. A piece of 1cm2 photographic film was dropped in. At this time the stop clock was started.

Conclusion: Trypsin works best at pH 9

Temperature experiment:

Apparatus: 7 test tube, thermometer, 2 beakers, 2 syringes, photo negative, scissors, trypsin, water with buffer solution, 5 water baths, test tube racks, stop clock

Diagram:

Method:

Firstly goggles don’t need to be worn but care needs to be taken with the water baths. I will keep this a fair test by using the same amount of a substance each time. The film was cut into seven 1cm2 pieces then put to one side. Each test tube was filled with 5cm3 of water and buffer solution pH 9 and 5cm3 of trypsin. Each of the seven test tubes was placed in one of the seven water baths with temperature range of 230 to 760 C. A piece of the film was placed in each test tube. At this time a stop clock is started. Each test tube is monitored until the film goes clear.

Results table:

Analysis:

The optimum pH of the enzyme was pH 9, which is what I predicted the preliminary experiment showed this. However the optimum temperature wasn’t 400 C but it was 710C, which is not what I expected this again goes back to the lock and key hypothesis. The higher temperature gave the active site become more elastic to change shape to fit the substrate. My conclusions are that trypsin works best at an alkali pH and a temp of 710 C.

Evaluating:

In my experiment I think that we should of used a bigger range of temperatures because then we may have been able to see when the enzyme denatured. I also think for the preliminary experiment we could have tested more buffer solutions to see whether the enzyme would have worked better in more alkali solution. I think that my results support my conclusion and there weren’t very noticeable anomalous.