Investigating the Factors That Affect the Activity of Trypsin

Planning

Aim: To investigate how pH affects the activity of trypsin.

Background Knowledge

An enzyme is composed of polymers of amino acids that act as catalysts within living organisms. They speed up chemical reactions within the body, without which these reactions would occur too slowly, causing the organism to die. They are not themselves used up in this process.

They break down large nutrient molecules within the body into smaller ones. The way they do this is by the lock and key theory. That is, each enzyme has its own shape that fits like a lock fits with a key with the appropriate substrate. It is the sequence of amino acids that make up an enzyme that determine the 3D shape of an enzyme and hence its specificity. Its unique shape prevents the enzyme reacting with the wrong substrate. An enzyme is a lot bigger than its substrate, so only a small portion, which comes into contact with the substrate, is active.

Enzymes usually (but not always) work best at neutral pH.

While heat does speed up a reaction, it can also change the shape of an enzyme (as can pH) making it useless.

The graphs show the effect of pH and temperature on an enzyme-controlled reaction.

As each enzyme has its own shape to fit its corresponding substrate, different enzymes are specialised for different reactions. For example, proteases, which break proteins down into polypeptides.

One example of a protease is trypsin. Trypsin is formed in the pancreas and then secreted into the small intestine where it aids the digestion of protein. Its optimum pH is 8.

Preliminary Results

(readings were taken using solution of pH 9)

Key Factors

pH – Enzymes are sensitive to changes in acidity/alkilinity and cannot work in extremes of either. Each enzyme has its optimum pH Trypsin’s is 8. The enzyme reacts to changes in the amount of H+ ions.
Temperature – Low temperatures can alter the shape of an enzyme, making it useless for the reaction it is specified for. High temperatures also denature the enzyme, rendering it inert.
Enzyme concentration – The higher the concentration, the faster the reaction will take place. This is because there is more of the enzyme available to break down the gelatine (which is the protein we will use).
Substrate concentration – In this case, it will mean a larger piece of photographic film (our source of the protein gelatine). If there is more gelatine to be broken down, it will take longer.

I have chosen to investigate how pH affects the rate of reaction with trypsin. This means the pH of the solution will be my independent variable.

Key Variables

pH – 3, 5, 7, 8, 9, 11
Temperature – room temperature (about 21°C)
Enzyme concentration – 5% trypsin (1ml)
Substrate concentration – 1cm x 1cm photographic film
Buffer volume – 3ml

Equipment

6 boiling tubes
tube rack
photo film
scissors and tweezers
goggles and protective gloves
buffer solution
5% conc. trypsin
syringes (1ml, 3ml)
stopclock
glass rod for stirring

Diagram

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I have chosen to investigate how pH affects the rate of reaction with trypsin. This means the pH of the solution will be my independent variable.

Key Variables

pH – 3, 5, 7, 8, 9, 11
Temperature – room temperature (about 21°C)
Enzyme concentration – 5% trypsin (1ml)
Substrate concentration – 1cm x 1cm photographic film
Buffer volume – 3ml

Equipment

6 boiling tubes
tube rack
photo film
scissors and tweezers
goggles and protective gloves
buffer solution
5% conc. trypsin
syringes (1ml, 3ml)
stopclock
glass rod for stirring

Diagram

Method

Set up the boiling tubes as shown in the diagram. Put 3ml of the appropriate buffer solution in each boiling tube, then 1ml of the 5% concentration of trypsin. Wear gloves and goggles, as the enzyme is irritant. Leave for a minute to equilibrate. Meanwhile, cut the photo film into 1cm x 1cm pieces, wearing gloves and using the scissors and tweezers, so skin doesn’t come in contact with the film. Add to the buffer solution and wait. Stir the solution at intervals, to loosen the silver particles from the film. When the film goes clear, the reaction has taken place.

Accuracy

In order to obtain enough evidence to support a firm conclusion, enough readings will have to be taken, to be accurate. The experiment will be repeated three times, to identify anomalous results and if found, will be re-tried. Ideally, we would like to test each pH one at a time, to avoid confusion, but we will be under time pressure so this may not be possible. So we may use a stopclock for each one. Another factor which may affect the accuracy of our results, is we may not know exactly when the reaction has taken place. Not only are 6 samples difficult to keep watch over, you can’t always tell if the reaction has taken place as there may still be loose particles which have not come free of the film. Another point to consider, is with the stirring. It is inevitable that one will get more vigorous stirring than another, it is impossible for humans to regulate their stirring exactly. This will cause loose particles of silver to come off, clearing the film sooner than otherwise, giving the false impression that the reaction took place faster than it actually did.

Prediction

From the knowledge I have obtained, I think that the reaction will be fastest at pH 8, as this is trypsin’s optimum pH. Those pHs closer to 8 will also react reasonably quickly but not as fast as 8, and the more acid or alkaline the buffer solution, the slower the reaction will be. This will be due to a deficiency/excess of H+ ions which will affect the enzyme’s performance. Looking at my preliminary results, the film took 419 seconds to go clear at 5% concentration under pH 9. Because this is slightly more alkaline (less H+ ions) than the optimum, I predict that the reaction at pH 8 will take slightly less than this. PH 10 and 6 should take longer, but both should have approximately the same rate as each other. PH 5 should take even longer and I do not expect pH 3 to react within any reasoable length of time.

The information in the Background knowledge was obtained from the following sources:

Encarta ’97 CD-ROM
Britannica.com

Cambridge Modular Sciences

Observation and Analysis

Results

Red indicates results obtained from a different source, under the same conditions.

Analysis

From looking at my graph, I can see that the pH at which the trypsin broke down the gelatine fastest was 8. At pH, the reaction took longest, and was not even measured at pH 3. This supports my original prediction, as I predicted that pH 3 would take far too long and 5 will take longer than pH 6 and pH 10. I did, however, in my original prediction say that the rate of reaction at pH 6 was 5.7, whereas at pH 10 it was a lot faster at 14, even though it is just as far in terms of pH from 8 as 5. From this, I can conclude that trypsin works better in alkaline solutions than in acid. The enzyme reacts to the amount of H+ ions or OH- ions. Too many of either will cause it to change shape, or become denatured, deeming it useless. If trypsin works best at pH 8, This means it works best in a slightly alkaline solution, i.e. the solution has slightly more OH- ions than H+. The graph shows that the enzyme doesn’t work as well with a greater presence of H+ ions; a steeper line on the acid side, indicating a sharper drop in rate. Meaning that trypsin does not work so efficiently in acidic conditions. Even at pH 7, which is neutral, the rate of reaction is 9.2, a significant drop from pH 8’s 19.1. This proves that trypsin works well under slightly alkaline conditions. I predicted that trypsin would work best at pH 8 and I predicted correctly. This information can also tell us something about the pH of the small intestine; that it is slightly alkaline.

Evaluation

I believe that the evidence obtained from my investigation is sufficient to support a firm conclusion. There is enough information to tell us that trypsin works best in alkaline conditions, and this can give us a hint as to the conditions in the small intestine. In order to test this, further investigation will have to be carried out to determine the pH of the small intestine.

We can see some anomalies, looking at the results. In the first instance, trypsin worked best at pH 9, whereas in the second it performed best at pH 8. There are many factors which could have caused this. The time we gave the buffer solution to equilibriate was not timed, and therefore not constant. Some were given longer than others and it is possible that perhaps pH 8 was not given long enough to equibriate in the first instance. Some pieces of photo film were also bigger than others, as it was difficult to measure them exactly and to cut them, holding them with the tweezers as we could not allow our skin to come into contact with the film. Another limitation was the stirring of the buffer solution. We tried to stir them all equally vigorously, however this was not always possible and some were shaken more vigorously than others, shaking some silver particles loose from the film, giving the false impression that the reaction took place faster than it actually did.

It is only by averaging the two results for pH 8 and 9 that we get the impression that 8 is the optimum. Although we know, from the background knowledge that it is 8, further investigation would be required in order to test this theory. Perhaps by investigating how the enzyme reacts under pH 8.5. Another thing would be to test each pH individually, as it was difficult to test them all simultaneously with one stopclock, as it was hard to tell exactly when all the gelatine had been digested. Ideally, we would have also liked to test each pH a third time, for greater accuracy. But overall, I think there is sufficient evidence to support a firm conclusion.