Investigation On The Enzyme Trypsin

Investigation on the Enzyme Trypsin

An Investigation determining a factor affecting the rate of digestion of gelatin by the protease trypsin.

Introduction
An enzyme is a biological catalyst, which speeds up reactions. An example of this in the human body is trypsin (a protease produced in the pancreas and used in the stomach), which catalyses the digestion of gelatine, a protein. For this investigation, a photographic film will be the source of the gelatine. I will be able to identify when the gelatine is digested, when the photographic film turns from a dark brown colour, to being transparent.

All enzymes are proteins, which are specific to the molecule that they break down. This is known as the ‘lock and key’ theory, where the active site only allows a specific substrate to be broken down, eventually resulting in easier absorption (larger surface area). Enzymes are made up of a long chain of amino acids, which form together in such a way as to leave a specific pocket, into which a substrate (as long as it fits perfectly into the pocket) can fit into it like a key in a lock (hence the ‘lock and key’ theory). The reaction then takes place, and the product of the substrate is then released. The enzyme, not changed by the reaction, can then perform the same “operation” on countless other substrates.

Because the enzyme can be re-used, only a small amount is needed. Despite this enzymes can make cell reactions go many million times faster than they would normally. Since enzymes are biological catalysts, by definition, they are not used up or changed in the reaction that they catalyse. Even though they cannot be used up, when subjected to a high temperature (50°C and above), enzymes can become denatured and the active site damaged or destroyed. After denaturisation, the enzyme becomes useless as no more substrates can become further digested by them.

Since there was ample trypsin for our use, and because trypsin begins to denature by 50°C (the temperature of the water bath I was using), I used a fresh batch of trypsin for each experiment I performed.

Before I started, it was important for me to decide what factor I was going to set as my independent variable and what I was going to setting as my dependant variable. There were several possibilities. Since speeding up the reaction was obviously one option, I could have changed either the heat of the reaction or the concentration of trypsin. This is because heat results in molecules moving faster, resulting in more particle collisions and a faster reaction (also each collision is more likely to contain the activation energy required for a successful reaction). Likewise, a higher concentration of trypsin would result in more trypsin particles being involved in the reaction, and there would be more chance of a successful collision. I took some preliminary reading to see if changing these variables might produce any interesting results.

Table showing the time taken for the trypsin to digest the gelatine, at varying temperatures (preliminary):

Time taken for reaction (seconds) Average Time taken for reaction
Temperature (°C) Reading 1 Reading 2 Reading 3 (Nearest second)

Table showing the time taken for the trypsin to digest the gelatine, at varying concentrations (preliminary):
Concentration Time taken for reaction (seconds) Average Time taken for reaction
Of Trypsin Reading 1 Reading 2 Reading 3 (Nearest second)

Another factor that could affect the rate of reaction is pH, because most enzymes become denatured by either very high or very low pH levels. In order to see at what pH trypsin was most functional, I did a preliminary experiment:

Table showing the time taken for the trypsin to digest the gelatine, at varying pH levels (preliminary):
pH level Time taken for reaction (seconds) Average Time taken for reaction
Of Trypsin Reading 1 Reading 2 Reading 3 (Nearest second)

As you can see my predictions were correct, increasing either the temperature or the concentration resulted in speeding up ...

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As you can see my predictions were correct, increasing either the temperature or the concentration resulted in speeding up the reaction, and the optimum pH level seems to be around 7. However, there was another variable that I suddenly became interested in. While taking the results above, I put the trypsin in the appropriate water bath at the same time as I put the film containing the gelatin into the trypsin. I wondered what would happen to the rate of reaction if I changed the incubation time of the trypsin, and for example heated the trypsin in the appropriate water bath for 1 minute before introducing the gelatin. To see if this would give any interesting results, I did another preliminary experiment:

Time taken for the trypsin to digest the gelatine, the trypsin having been incubated for varying amounts of time (preliminary):

Incubation time Time taken for reaction (seconds) Average Time taken for reaction

Of Trypsin (seconds) Reading 1 Reading 2 Reading 3 (Nearest second)

This shows no clear trend, so I therefore decided to further investigate what effect changing the incubation might have on the rate of reaction. This would mean keeping the temperature, concentration of trypsin, and the pH of trypsin (the other independent variables) the same throughout. This was required in order to make it a fair test, so that the only thing that was being altered was the incubation time. I would measure the time taken for the gelatin to be digested by the trypsin, and record all of my results in a table.

I then needed to decide what concentration of trypsin and which water bath to use. My preliminary results on varying concentration showed that a 3% solution of trypsin digested gelatine at a moderate rate, and since I didn’t want the digestion to take either too long or too short, I used a 3% solution of trypsin in my investigation. My preliminary results on varying temperatures show that trypsin heated in a 50°C water bath also digests at a moderate rate, and since I knew that at 50°C trypsin would be denaturing to an extent, I decided to use a 50°C water bath in which I incubated the trypsin before introducing the gelatine.

Hypothesis
I predict that a given constant proportion of the functional trypsin present in a solution at 50°C, will decay and denature every minute (i.e. exponential decay).

I think that trypsin at a temperature of 50°C has a certain chance of survival. In the first couple of minutes, the rate of reaction will in fact have increased, because the amount of denatured trypsin would not compensate for the fact that the temperature had risen, so the molecules would be moving faster, hence more collisions would take place (and more achieving the activation energy required), and this would result in a faster rate of reaction. Also, when in the water bath, the trypsin took about 3 minutes to warm up to 50°C, so before this the rate would rise, because not much denaturisation would have taken place, and all the molecules would be moving faster, speeding up the reaction. However, as more and more trypsin became denatured, the amount of denatured trypsin would become a more significant factor than the faster moving particles. So, after a few minutes, the rate of reaction would begin to decrease, and I think that the rate will decrease from this point onwards by a constant proportion every minute. For this reason I expect my graph to look something like this:

Method
In order to perform my investigation, I used the following apparatus:
• Trypsin (pH 7, 3% concentration)

• Photographic Film (gelatine source)

• Water Bath set at 50°C

• Thermometer

• Test Tubes

• Wooden Splints

• Syringe

• Stopwatch (accurate to 1 second)

Throughout my investigation, all that I changed was the incubation time of the trypsin, and I made sure that the other independent variables did not alter. This was in order to make the investigation as fair as possible, and so my results could be as accurate as possible.

A 50°C water bath was prepared to the nearest °C, and I checked with my thermometer that the water was indeed at 50°C. I then prepared my gelatine, by sliding the photographic film inside the end of the wooden splints. This was done so I could easily operate the gelatine source, and it was much easier to control when it was on the end of a wooden splint. Then I used the syringe to fill a test tube with 2cm3 of trypsin. This was a convenient level, as half of the photographic film would fit inside the trypsin, and I could easily tell when the gelatine had been digested, by comparing the film that had been in contact with the trypsin, and the other half of film, which had not.

Once the test tube was prepared, I placed it in the 50°C water bath, and I started the stopwatch. I left it in there (on its own – with no photographic film) for however long I wanted to, and when that time had been reached, I placed the splint containing the film into the test tube. At this point I restarted the stopwatch, and I checked the gelatine for any signs of digestion every 10 seconds. When I realised the photographic film would soon go transparent, I kept a closer eye on the film, and checked every couple of seconds. This way, I was able to record my results pretty much accurately to one second. I noted down the result, and repeated the exact same experiment (with the same incubation time) 5 times. After taking 6 recordings for that incubation time, I then repeated the experiment but increased the incubation time by 1 minute, up until I had obtained results for an incubation time of 20 minutes.

I took 6 readings for each incubation time to help increase my accuracy, and identify anomalous results. If I had, for example, by mistake used the wrong concentration of trypsin, the effect of this result would be heavily diluted, so in the end it would not have made much difference to the average rate of reaction for that incubation time. I used 21 different incubation times, ranging from 0 minutes to 20 minutes, which I thought gave me a sufficient range of results to accurately analyse, and draw conclusions from.

Throughout the investigation, while performing any kind of experiment, I wore safety glasses, in order to ensure that no hot water (some water baths were at 80°C) or chemicals (i.e. trypsin) came into contact with my eyes. (In case of an unexpected fire, a fire extinguisher was handy and there were 2 exits to the lab).

Results
I recorded my results, and I then tabulated them. I also constructed a graph, which can be seen on the next page.

A Table showing the rate of reaction for trypsin digesting gelatin, the trypsin having been incubated for varying amounts of time.

Incubation time (minutes) Reading 1(seconds) Reading 2(seconds) Reading 3(seconds) Reading 4(seconds) Reading 5(seconds) Average time for Trypsin to Rate (4 d.p.)
dissolve (seconds) (1/average time)

Analysis/Conclusion
It seems at first glance that my prediction graph (on page 4) is accurate, when compared to my graph of best fit (on page 7). Although I had predicted that the rate of reaction would increase during the first few minutes, I was somewhat surprised at how much the rate had increased. By the way things look on the graph, it seems as if the rate starts to settle down to a constant decrease, by about 5 minutes of incubation time. For this reason I decided to concentrate on the graph from 5 minutes and onwards, since before this point the trypsin had not reached 50°C. For each minute from 5 onwards, I found out what percentage of trypsin had denatured, by working out the percentage decrease in the rate of reaction. Here are my results:

A Table showing the relationship between the Incubation Time period, and the % decrease in the rate of reaction
Incubation Time (minutes) % Decrease in rate of reaction

So, since the average percentage decrease between incubation times is 3.8%, that suggests that on average 3.8% of the functional trypsin becomes denatured every minute that it is at 50°C. The reason for the expediential decay is that when at 50°C, trypsin has a certain chance of survival. My results suggest that on average, the chances of a trypsin molecule becoming denatured during a minute at 50°, are 3.8%. Another way of putting this is that 3.8% of the functional trypsin present in a solution at 50°C, will decay and denature every minute.

Sources of Error
I think that, since I took 5 readings for each incubation time, my accuracy was very high. However, if there was a serious fault (for example if I by mistake used 2% trypsin instead of 3% trypsin), the chances are that all the readings were affected. If you look at the table above, there is a strange trend in that from 10 – 14 minutes of incubation time, the decrease in rate was much lower than the other readings. I think that, since I performed those readings all on one day, either I was supplied with the wrong percentage or pH of trypsin, or I made a mistake when filling my beaker with trypsin. I certainly think that there was some kind of mistake made on that day, and I think that explains the anomaly. I also had some difficulty with telling when the entire gelatin had been denatured. The film turned from opaque brown to clear, but it was hard to tell exactly when this transition took place. For this reason, I decided to remain consistent when taking my readings, and I stopped the stopwatch as soon as I could see through the film at all. What also caused some problems was the timing. It was impossible to start the stopwatch and put the splint in at exactly the same time, so I ended up putting the splint in about one second after the stopwatch was started. However, this happened for pretty much every reading, so at least relative to each other, my results remained quite accurate.

One other factor that might have had an affect on the results, is that fact that trypsin does denature (although very slowly) at room temperature. For this reason, the percentage of functioning trypsin would depend on how long it had been since the trypsin had been manufactured. As long as all the trypsin we were using had been manufactured at the same time, this would not have been a serious problem.

Evaluation
Although my hypothesis was quite accurate, there were several inaccuracies. Despite the obvious fact that a few of my readings were subject to the wrong concentration of trypsin (I investigated further into this matter and, as it happens, I was using 2 % trypsin on this day instead of 3% trypsin), my results were quite poorly matched. My table shows significant variation in percentage decrease, and I think that this was mainly due to timing difficulties. There was also the problem that when I took the splint out to check on the film, for that short period of time the film was not in contact with the trypsin, although the stopwatch was not stopped. Since I had to take each splint out several times to check on the progress of the denaturisation, this built up and would have resulted in at least a few seconds of inaccuracy for each reading.

For this reason, if I were to perform the experiment again, I would have asked a classmate to help me when placing the splints in the trypsin. I would ask them to start the stopwatch at exactly the same time as I placed the splint in the trypsin. Also, I would have done 2 experiments at a time. One of them I would check frequently and the other I would leave untouched. When I saw that the one I was checking on had almost gone clear, I would then check the other one and stop the stopwatch when that one had gone clear. I would do this to reduce the amount of time that the gelatin spent outside the trypsin, and I would ignore the result of the other experiment. This, I feel, would improve the accuracy of my experiment, and would probably supply more constant results.

In order to extend my investigation, I would investigate the denaturisation of trypsin at 60°C, and see if expediential decay still occurred. I would first wait until the temperature had reached 60°C (this would take a few minutes), and then start timing, because before the trypsin reaches 60°C, the results produced cannot be formed into a conclusion analysing trypsin at 60°C, as the trypsin would never have reached 60°C. (This also leads into the problem that waiting a few minutes for the trypsin to warm up would result in some of the trypsin becoming denatured before timing had started).

Investigation On The Enzyme Trypsin

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