We are going to find out how different enzymes (in this case, trypsin) concentration in a solution would affect the rate of which solute (Casein) is broken down.

by kinyung102902hotmailcom (student)

Enzyme concentration and enzyme activity

For this investigation, we are going to find out how different enzymes (in this case, trypsin) concentration in a solution would affect the rate of which solute (Casein) is broken down. I think enzyme concentration would definitely have an effect on the rate of reaction. My hypothesis is that increasing the concentration of trypsin would also increase the rate of catalysation of casein.

Enzyme (a globular protein) is used to catalyse specific biological reactions by lowering the activation energy and they are left unchanged by the end of the reaction, for example trypsin is responsible for catalysing the break down of casein (a protein typically found in milk - it is what makes it ‘opaque’) into smaller molecules which the body can absorb. This means the reaction can occur in a faster pace. This is important since without enzymes the molecule cannot break down fast enough on its own to provide us with constant nutrients.

The reaction happens with an enzyme-substrate complex forming between trypsin and casein.The formation of this complex is explained by the induced- fit hypothesis,where the enzyme has a distinct and flexible active site, as oppose to a rigid one suggested in the lock-and-key model. Once the casein enters and attaches to the active site of trypsin, the shape of its site will modify around the substrate to form the active complex, once the reaction is complete, the now broken down substrate will leave the active site and the enzyme reverts to its previous, unmodified form. This helps to explain why enzyme are so specific that they only catalyse one reaction.

Rate of reaction of enzyme is directly linked to enzyme concentration, (given that the factors which affects enzyme activity remains constant at a point where trypsin is at active condition) as concentration of enzyme increases, so does the rate of reaction. This is due to the increased possibility of enzyme colliding with a substrate to form the enzyme-substrate complex (because there are more enzyme molecules present in a given value.).

Independent variable in this investigation is the concentration of trypsin (2%, 1%, 0.5% and 0.25%) as that is what we will be changing, whereas the dependent variable would be the rate of reaction since that is the value we will be measuring.

The control variable (anything else that can influence the dependent variable) include the following, note that they should all be kept at the most optimum conditions possible in order to carry out a fair test in the sense that the enzyme concentration should be the limiting factor to the rate of reaction (and at the end the substrate concentration):

Temperature: The trypsin solutions are stored at its optimum temperature (right up until we take it out for the reaction) before the investigation, which is around 37oC (body temperature). Controlling temperate level is important since it is one of the factor which affects enzyme activity.

If temperature is <5oC, the enzyme is inactive, while if temperature is >40oC, it starts to become denatured and at 60oC, enzymes would be completely denatured. In both cases it means the enzyme can no longer carry out its function. Inactive state is when the enzyme still retained the shape of its active site but can no longer catalyse any reaction, whereas denatured means the enzyme has unravelled and completely lost its active site, and therefore cannot return to its previous shape.

pH: Enzymes work best within their pH range, and trypsin’s optimum pH range is about 7.5-8.5, ...

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pH: Enzymes work best within their pH range, and trypsin’s optimum pH range is about 7.5-8.5, which is about neutral (if slightly alkaline) so it isn’t too hard to achieve. The effect pH has on trypsin is similar to that of temperature, as pH move further off the optimum pH range, the rate of reaction would decrease (a enzyme starts to denature), but if extreme pH are reached then the enzyme would become completely denatured and can no longer carry out its function.

Substrate concentration: Substrate concentration affect rate of reaction in a similar way to enzyme concentration, however, since we’re instigating the effect of different concentration of enzyme, we must keep this variable constant - therefore we will be using 4% casein solution. As substrate concentration increases, rate of reaction increase as there is more chances of the trypsin and casein colliding to form the enzyme-substrate complex.

Volume of solutions added: Since the cuvette has a maximum capacity of 4 cm3, we have decided to use 2cm3 of trypsin solution with 2cm3 of casein solution, this must be kept constant as we’re only finding out the affect of enzyme concentration on rate of reaction, and amount of solutions added may affect that - i.e. if volume of enzyme solution increased, amount of active site available for catalysation would also increased. Having more than 1 independent variable would make the results unreliable since we would not know which factor was affecting the end result.

For this investigation, we will be using the following apparatus and chemicals.

Equipments:

- Colorimeter (& cuvettes)

- Stop clock

- 5cm3 Syringes

- Test tubes

Chemicals:

- 4% Casein solution

- 2%, 1%, 0.5% and 0.25% Trypsin solution (kept at 37oc)

- Distilled water

To investigate how enzyme concentration affects rate of reaction. First of all, we needed to calibrate the calorimeter, we filled a cuvette with 4cm3 (this is the maximum capacity the cuvette can hold) of casein solution using a syringe, and set the absorbancy to 0.5, because it isn’t possible to set it to 0. Also, since absorbancy would decrease as the casein progresses to be broken down, setting it to 0 would just make it into a negative value.

Secondly, we obtained 2 cm3 of casein with a syringe and put it in a test tube, then we also added 2cm3 of distilled water into the same test tube (using a different syringe to avoid contamination). Shake the test tube and start the stop clock straight away. After that, we immediately pour the mixture into the cuvette, doing so quickly (before the 1 minute mark hit) since we have to take the reading of the initial absorbancy.We put the solutions into a test tube instead of directly into the cuvette because we should shake the solutions to ensure all of them has reacted, whereas it would be hard to mix them together in a small container like a cuvette. This would be our control (0%), this would allow us to know what happens if there is no trypsin present to catalyse the break down of casein.

Our team has decided to take the absorbancy reading every minute for 10 minutes since it seemed like an appropriate time interval, 30 seconds seems to rushed and we may not be able to see a clear pattern of absorbancy if we take the reading more than 1 minute apart. Furthermore, we’ve decided to set the observing period as 10 minutes because rate of reaction seems to start to even off by that time and so we would not need to observe any longer.

We then discarded the content in the cuvette and rinsed it with distilled water repeated all the steps from adding 2cm3 of casein for each of the remain concentration of trypsin instead of using distilled water. We had to rinse the cuvette to make sure the solution left over would not affect the result of our next concentration.

Apart from all the precaution we’ve set up in our investigation, there are still errors made as shown in our original result (very inaccurate, the values increased rapidly instead of decreasing), we could have taken more care to improve our method. These improvements will be mentioned and explained later on. (The results on the next page are ones we obtained from our class since our own results are too inaccurate to use.)

The class result:

The results shows there is definitely a decrease in absorbancy as we decrease the trypsin concentration at a certain time interval. This means there’s a decreased rate of reaction as we reduce enzyme concentration. This is due to casein being the protein which cause the solution to look milky, so when the casein is being broken down, the solution turns clear. What we are measuring here is how quickly the solution is turning clear.

As you can see from the graph, at 0% (with distilled water instead of trypsin) there is no change in absorbancy, meaning no reaction is occurring or that it is happening very slowly. Since enzymes do break down on its own over a long period of time (as their bonds weaken), perhaps we would observe a reaction if we measure absorbance for a longer time frame, however due to limited time, this was not possible.

As you can tell at 0.25% of trypsin, there are signs of reaction, however it is rather slow, this is due to there being not many enzyme molecules in a certain volume, making it less likely to collide with a protein to form an enzyme substrate complex. Also, there is a lot of substrate to be broken down compared with the amount of enzyme present, so it would take a longer time to break down all the casein molecules. Form the graph it seems that the absorbancy readings are similar for 0.5% and 1% as well, perhaps because we are increasing enzyme concentration at a slower rate than from 0% to 1%, therefore there are not as much difference in change in absorbancy than 0.25%.

At 2% we see solid evidence how enzyme concentration affects rate of reaction, at 1% the absorbance decreased by 0.1 (0.50 - 0.40 = 0.10) in 4 minutes, whereas at 2%, the absorbancy decrease by 0.32 (0.50 - 0.18 = 0.32) in 4 minutes, this shows the average initial rate of reaction for 2% is almost 3 times as much as that for 1%. Also, after 4 minutes, rate of reaction has completely leveled off.

This is due to the fact that the more enzyme molecules in a solution, the more likely a substrate molecule will collide with one and bind to its active site to from an enzyme-substrate complex. However if the amount of substrate is limited, then there will be a point where increasing enzyme concentration will have no further effect as the rate of reaction is no longer limited by shortage of enzyme but by other factors (i.e. Concentration of casein).

There are a few improvements which we could make for this investigation to be more effective. In our original set of results, the value of absorbancy kept increasing over time and it even exceeded the calibrated absorbancy of 0.5. This should not happen since casein solution should gradually be turning clear and so absorbancy should decrease. Also exceeding 0.5 means the solution is turning even more opaque than the actually casein solution, which isn’t possible. The main reason for this could be faulty calorimeter, because previous mistake could not have caused this huge inaccuracy. I think one way to avoid it is to perform a preliminary trial prior to the actual investigation in order to spot any fault in the apparatus or chemicals by comparing the result to those posted online / in journals from reliable sources.

Also, during the investigation, we should check whether the absorbancy is the same every time the calorimeter is empty to see if we used a consistent calibration. Otherwise, the knob on the calorimeter could’ve been accidentally moved or the calorimeter could be faulty and we would know about it until much later in the investigation.

Furthermore, since we have only allocated one syringe for casein and one for trypsin, we’ve had to use the same syringe for every concentration of trypsin, and since we haven’t thought of rinsing it every time we use a different concentration, the result may have been affected due to this. Next time it would be best to use a different syringe fr each concentration to avoid further contamination.

Additionally, we could also extend the time range to perhaps 15 minutes to see if the absorbancy values stayed constant or keep decreasing very slowly. This will allows us to see a little beyond what we know now and we can deduct a better conclusion from it.

Lastly, the fact that we took out the trypsin solutions much earlier than we needed them might have has a effect on the rate of reaction as trysin works best at 37oc but as it leaves the water bath, it would starts to decrease towards room temperature (around 18 - 21oc), therefore temperature could actually be a limiting factor here too, because the lower the temperature the slower the rate of reaction. Next time, I would suggest we perform and experiment close to the water bath so we can extract the trypsin whilst it is still inside the water bath - hence there will be less time for it to cool down to room temperature.

This class result has proved my hypothesis and prediction that as enzyme concentration increase, so did the rate of reaction. Despite the fact that a few improvement could be made to make the method more effective , the step we followed did consider most of the aspect which could’ve made the results unreliable and inaccurate. We used precautions to try and reduce the amounts of errors made. However, in the end the calorimeter we used (to produce our set of result) seemed to be faulty, therefore there wasn’t really anything we could’ve done to avoid it apart form early detection (of faulty apparatus) by performing a preliminary trial. Overall I would say this is not a very successful investigation within our team since looking at our own result, it did not prove my hypothesis. It was only when looking at the class results was I able to make correct deduction and analysis.

Risk assessment

Reference/ image sources

pH graph

http://alevelnotes.com/Factors-affecting-Enzyme-Activity/146

Enzyme - Substrate complex diagram http://www.hsc.csu.edu.au/biology/core/balance/9_2_1/921net.html

Enzyme concentration graph

http://alevelnotes.com/Factors-affecting-Enzyme-Activity/146