Sie sind auf Seite 1von 17

# IB Diploma Biology

IA TASK 3I The Effect of Temperature on the Clearing of Film Negative by the Enzyme Trypsin

## Eugene Hui 12.5

Data Table: Time Taken for Trypsin to Digest Gelatin across Six Different Temperatures
Temperature (0.5C) Actual Temperature (0.5C) Duration for Trypsin to Clear Film Across Various Temperatures (0.5 seconds) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Duration Duration Duration Duration Duration Duration Duration Duration Duration Duration in in in in in in in in in in Minutes Seconds Minutes Seconds Minutes Seconds Minutes Seconds Minutes Seconds Average Duration in Seconds Average Duration in Minutes (0.5 seconds) Standard Deviation of Average Duration in Seconds (seconds and % uncertainty, respectively) 3.51, 0.66 15.0, 3.37 1.59, 0.95 20.3, 5.34 13.7, 3.04 134, 14.5 Rate (seconds-1) Standard Deviation of Rate (% uncertainty)

40 45 50 55 60 65

39 45 49 54 57 63

## 0.66 3.37 0.95 5.34 3.04 14.5

Additional Notes: All data is recorded to three significant figures (with the exception of duration in minutes for trials held at 65 degrees, which have been recorded to four significant figures). Actual Temperature refers to the water bath temperature prior to the start of each trial. Absolute uncertainty was not expressed for rate, as the value would be too small to have any significant meaning. Columns with blue headings indicate raw data; yellow indicates processed data. Observations/Qualitative Data: Solution gets cloudy and purple while forming a dark colored precipitate as the film begins to clear. Film originally starts off being black, but turns purple during the process of digestion by trypsin. Film starts clearing from the center, with all corners being the last to be cleared.

Page 2 of 17

Eugene Hui 12.5 Sample Calculations: 1. Duration in Seconds To facilitate a smoother process when graphing results, all duration in minutes should be converted to only seconds. This can be done as follows: = 60 + For example, to convert 8:54 minutes to seconds (trial one at 40 degrees), the following needs to be done: = 8 60 + 54 = 534 With this, the process simply needs to be repeated for all trials at all temperatures. 2. Average Duration in Seconds While it is useful to know how long each trial took to clear the photographic film at a given temperature, the average duration at a certain temperature is equally important. As mentioned previously, seconds rather than minutes will be used as the standard measurement unit of time, so average should also be expressed in terms of seconds. The average is easily calculated, using:
(. ) = (. )

For example, to calculate the average duration in seconds for the 50 degree trials, numbers need to be plugged in as follows: (. ) = 274 + 275 + 269 + 271 + 270 = 271.8 5

But since all data is being expressed to three significant figures, this would mean that the average duration to clear the film negative in the 50 degree water bath was 272 seconds.

Page 3 of 17

## Eugene Hui 12.5

3. Average Duration in Minutes In order to have another set of data as reference, it would be quite useful to calculate the average duration of film clearing at each given temperature. Again, the duration in minutes will not be graphed. Average duration in minutes can be obtained through:
(. ) = (. )

## Using the figures again for the 50 degree trials:

(. ) = 4: 34 + 4: 35 + 4: 29 + 4: 31 + 4: 30 22: 39 = = 4: 31.58 5 5

Once rounded to three significant figures, this would therefore indicate that the average time it took for trypsin to clear the film negative at 50 degrees was 4:32. 4. Rate Rate is usually defined as the amount of product formed per unit of time. For this lab, the amount of product is simply represented by the clearing of the film negative. Clearly, the amount is not quantifiable. However, it is possible to assume that the amount of product is equal to one (1), and use this assumption to establish rates across different temperatures. Note that this rate merely pertains to this set of data, as it has no statistical value outside of the lab. In other words, the rate is simply a set of numbers with a somewhat arbitrary unit used to compare how fast or slow trypsin was able to clear the photographic film in each temperature. This can be done by diving the amount of product formed (1) by time taken:
( 1 ) = 1

For example, the rate for the 50 degree trials can be obtained through:
( 1 ) = 1 = 0.00367647 1 272

Rounded to three significant figures this would indicate a rate of 0.00368 seconds-1. As mentioned previously, this is just a number to compare the different rates within this lab, but bears little scientific meaning as it has no units strictly speaking. Furthermore, this statistic plays a more pivotal role in graphs, but less in the processed data table itself (for more information, please see graphs).

Page 4 of 17

## Eugene Hui 12.5

5. Standard Deviation of Average Duration in Seconds Standard deviation is a figure that indicates how much individual values deviate or spread from the mean, which would be the average diameter or rate of digestion. Standard deviation is helpful because the mean only indicates the middle value, but does not reflect the real spread of a set of data. (Standard Deviation, n.d.) Standard deviation is a bit more complex to calculate, but can be derived through: ( )2 = 1 Where M is the mean, X is each value, and n is the number of values. To calculate the standard deviation for all values pertaining to the 65 degree trials for example, it might be helpful to calculate all (X-M)2 values first before plugging into the formula. Note that units can be ignored. This is shown as follows: Trial 1: (X-M)2= (929-810)2=14161 Trial 2: (X-M)2= (929-851)2=6084 Trial 3: (X-M)2= (929-890)2=1521 Trial 4: (X-M)2= (929-940)2=121 Trial 5: (X-M)2=(929-1153)2=50176 After all values of (X-M)2 have been calculated, they can be plugged into the formula to calculate the standard deviation.
14161 + 6084 + 1521 + 121 + 50167 = = 18013.5 = 134.214 51

In this case, this number (rounded to 134) shows that the time taken to clear the film negative at this particular temperature tends to deviate from the average (929 seconds) by around 134 seconds in either direction. This is also called the absolute uncertainty.

Page 5 of 17

## Eugene Hui 12.5

6. Absolute to Percentage Uncertainty For purposes of convenience, absolutely uncertainties should be converted to percentage uncertainties. This can be done by:
% = 100

For example, using the same absolute uncertainty calculated in the previous step, the percentage uncertainty would therefore be:
% = 134 100 = 14.4241119% 929

Rounded to three significant figures, the percentage uncertainty for the average duration in the 65 degree trials would be 14.5%. Note that the percentage uncertainty is the exact same for the rate. This is because rate is equivalent to the reciprocal of average duration in seconds, therefore percentage uncertainty gets carried over and remains unchanged. But of course, because rate is so small, there would be no logical reason to convert percentage uncertainty to absolute uncertainty for rate, as the uncertainty would be too small to make any scientific sense in our current understanding. Science Behind the Experiment: Enzymes are globular proteins that speed up a specific chemical reaction, but remain unchanged in the reaction. They are specific, meaning one enzyme can only catalyze one reaction. However, they are also reusable, thus they also exist in small quantities. In addition, enzymes have an active site, which is responsible for the binding of a particular substrate. Once a substrate binds to the active site of an enzyme, a metabolic reaction occurs which ultimately results in the breakdown of the substrate into products. The complex formed during this process is called an enzyme substrate complex. In humans, trypsin is an enzyme excreted in the pancreas and takes part in food protein digestion, among other biological processes. Often at times, it is given to those who lack enzymes needed for proper digestion. (Trypsin, n.d.) Meanwhile, gelatin is a protein made from animal products, often involved in the preparation of foods, medicines, and supplements. With this, trypsin can break down this protein in the human body. (Gelatin, n.d.) In this lab, gelatin is the substrate, while the enzyme is trypsin. While the digestion of gelatin on photographic film will release silver halides that cause photographic film to clear, the rate at which this happens is also highly susceptible to the surrounding environments temperature. There are also other factors affecting enzyme activity, including substrate/enzyme concentration, pH, and the presence of inhibitors. Page 6 of 17

## Eugene Hui 12.5

By submerging a piece of photographic film in a 5% trypsin solution, the surrounding trypsin will be able to slowly digest the gelatin on the film, ultimately causing the film to turn clear over time. As temperature is the independent variable for this lab, it is clear that there will be a large range in time taken for the film to clear. That being said, temperature involves a concept of denaturation within enzymes (explored in the next few sections). Denaturation is the process in which temperatures above an enzymes optimum temperature affects the stability of the tertiary and quaternary structures. Essentially, bonds begin to break, and a shape change in the active site ensues. This process is usually irreversible, and goes forth to affect rate. Denaturation can be summarized briefly in this picture below:

Page 7 of 17

1200 1000

## Time Taken (Seconds)

800 600 400 200 0 40 Degrees 45 Degrees 50 Degrees 55 Degrees 60 Degrees 65 Degrees

Temperature (Celsius)

The graph above plots the average duration in seconds for the photographic film to be cleared across different temperatures. Note that standard deviation exists for all data points, but only the 65 degree data point has a large standard deviation, thus the large error bars. Figure 2:

## Rate of Gelatin Digestion by Trypsin in Different Temperatures

0.00400 0.00350

Rate (seconds-1)

0.00300 0.00250 0.00200 0.00150 0.00100 0.00050 0.00000 40 Degrees 45 Degrees 50 Degrees 55 Degrees 60 Degrees 65 Degrees

Temperature (Celsius)

This graph indicates the rate at different temperatures. In essence, this graph simply is a mirrored image of the previous graph, but has crucial information nonetheless.

Page 8 of 17

## Eugene Hui 12.5

Conclusion: Based on the results seen in figure 1, it can be seen that temperature does not form a linear rate with time taken for trypsin to clear the film negative. Using the trend line alone, the general idea seems to point towards a notion that time will reach a minimum, before increasing once again when temperature is either too high or too low. For example, the average time for the 40 degree trials was 533 seconds (8:53 minutes), while in the 45 degree trials the average time dropped to 447 seconds (7:27 minutes). The lowest time occurred at 50 degrees, which took only 272 seconds (4:32 minutes) on average. Any temperature higher than this results in longer average times, such as in the case of 55 degrees, which took 382 seconds (6:22). By looking at the trend line as well, it can be seen that the vertex lies at around 50-51 degrees Celsius, indicating that the lowest time taken for trypsin to clear the film negative should be within that particular range. While figure 1 is useful in indicating how duration in seconds decreases up to 50 degrees, then rises thereafter, it is also useful to look at rate itself. Assuming that the original observations drawn from figure 1 alone were correct, then figure 2 should corroborate the same findings as well. For example, in the 40 degree trials, rate was at 0.00187 seconds-1. By 50 degrees, rate had increased from 0.00224 seconds-1 at 45 degrees to 0.00368 seconds-1. Furthermore, decreases in rate after 50 degrees are clearly demonstrated by the subsequent rates of 0.00262 seconds-1, 0.00222 seconds-1, and 0.00108 seconds-1 for the 55, 60 and 65 degree trials, respectively. Based on the findings of figures 1 and 2, it can be seen that 50 degrees is roughly the ideal temperature which brings forth the greatest amount of activity within trypsin. On the other hand, duration in seconds was longer and rate were lower at 40 and 45 degrees, likely due to the fact that the substrate and the enzyme did not possess enough kinetic energy to allow for enough successful collisions at lower temperatures. At lower temperatures, the substrate and enzyme would have fewer collisions at lower energies, bringing about fewer collisions that form a substrateenzyme complex. In the physical world, this would reflect in a slower rate of clearing of the film negative. As temperature increased, however, the substrate and enzyme would collide more frequently and at higher energies, thus substantially more substrate enzyme complexes were formed in the same period of time. In the case of 50 degrees, a higher temperature would have directly caused a greater rate and a lower time to clear the film negative. An interesting deduction from figure 2 also shows that a ten degree increase in temperature will generally yield a two-fold increase in rate. Of course, this is all under the assumption that 50 is the ideal temperature to achieve the highest rate. For example, in the 40 degree trial, rate was at 0.00187 seconds-1, but at the ideal temperature of 50 degrees, the rate had increased to 0.00368 seconds-1. By dividing the two rates (0.00368/0.00187), it can be seen that the ratio is roughly 1.97, which evidently demonstrates the idea that ten degree increases up to the ideal temperature will bring forth a double in the rate. Page 9 of 17

## Eugene Hui 12.5

While it is often assumed that increasing temperature indefinitely would cause an indefinite increase in rate, this is not the case for this lab. With enzymes, a key concept to take into consideration concerns denaturation. As mentioned previously, denaturation occurs in an enzyme when surrounding temperatures are too high and begin to permanently alter the shape of the active sites. Once trypsins active site changes shape, gelatin can no longer bind, causing a substantial increase in time taken. Furthermore, lab data shows that denaturation is not an instantaneous process. Even though it is clear denaturation occurred, the fact that the film negatives eventually cleared indicates that some active sites still remain unchanged. For example, rate at 55 degrees dropped to 0.00262 seconds-1, and at 65 degrees it was merely 0.00108 seconds-1. As rate was not at zero, this would have indicated that digestion was still occurring, albeit at a much slower rate. With this, it is likely that increasing temperature past 65 degrees will only worsen the state of denaturation in trypsin. A higher temperature would allow gelatin and trypsin to have greater energy, allowing for more successful collisions that result in the formation of substrate enzyme complexes. Ultimately, these complexes allow for the gradual clearing of film negative. At the same time, it is clear that beyond a certain temperature threshold, trypsin would also undergo denaturation, where certain molecules of trypsin would be rendered useless due to denaturation. In other words, any lower temperature higher or lower than 50 degrees would take a longer time with a lower rate. With this, the lab clearly shows that 50 degrees is the ideal temperature for trypsin that would bring forth the greatest rate and the least time to clear the film negative. Existing Experiments: The results of this lab evidently highlight the concept of an ideal temperature, also known as an optimum temperature for enzymes. Any temperature higher or lower than the optimum would bring forth a decrease in the total enzyme activity.

(Enzymes and Inhibitors, 2010) This conclusion is clearly supported across various sources, including the diagram to the right. For example, human enzymes have an optimum temperature is around 40 degrees (37 degrees to be precise), in which any temperature above that would immediately decrease enzyme activity due to denaturation. A lower temperature would cause lower enzyme activity levels, since there are fewer successful collisions that form substrate enzyme complexes. While the diagram partially highlights this concept, it is also a wellestablished fact that a ten degree increase in temperature will result in rate to double (up to the optimum temperature), corroborating this labs findings again. (Enzymes and Inhibitors, 2010)
Page 10 of 17

## Eugene Hui 12.5

(Effect of Temperature on Trypsin Activity, n.d.) In another lab, the optimum temperature of trypsin was found to be at around 37 degrees. Note that the trypsin used was human trypsin, whereas in this lab the trypsin used was not human trypsin, and would therefore have a different optimum temperature. Nonetheless, this corroborates this labs data in the sense that rates were lower at temperatures higher and lower than the optimum temperature. Furthermore, decrease in rate at temperatures above the optimum were attributed to denaturation in the aforementioned lab. This also supports this labs findings in the sense that denaturation is shown to be a gradual process as temperature continues to increase. Because the origin of trypsin in this lab remains rather obscure, it is hard to verify whether the optimal temperature observed from this lab is indeed correct. Despite there, there is irrefutable evidence that the general findings of this lab are indeed correct.

Page 11 of 17

## Eugene Hui 12.5

Reliability of Data: As a whole, data from this lab yielded very desirable results that clearly highlighted how temperature played a role in the clearing of film negative by trypsin. As far as data goes, the most obvious anomaly concerns the data point for the 65 degree trial. For both rate and duration in seconds, the percentage uncertainty was at 14.4%. As percentage uncertainty (standard deviation) is a measure of spread of data, it is not surprising to see that trials took anywhere between 800-1100 seconds. While at first this could suggest that an error occurred at this temperature, this could also be an indication of the severity of denaturation, as such high temperatures have all caused varying numbers of trypsin to have their active sites change shape. But as it is not known what happened, trials at this temperature should be repeated again. Theoretically, if rate is said to double for every ten degree increase in temperature up to the optimum temperature, then the rate at 45 degrees should have been the average of the 40 and 50 degree rates. But in this case, rate fell short at 0.00224 seconds-1, when the average was supposed to be at 0.00278 seconds-1. Because of this, it is not surprising that the data point was also below the trend line in figure 2. Such anomalies are hard to identify initially, as they only appear as anomalies once data all data has been processed. However, given the fact a clear conclusions were able to be drawn, it is highly unlikely that these anomalies significantly affected the final outcome of the lab.

Page 12 of 17

Eugene Hui 12.5 Evaluation of Procedure and Improvements: There were many variables that could have potentially limited the lab itself. Below is a table listing many of the limitations and their effects on the lab. Please note that the factors are ordered from most to least significant. Limitation Clear not defined Effect of Limitation The objective of the lab was to see how temperature affected the time it took to turn the film negative clear. However, clear was not defined, and hence, every trial was most likely stopped at a different stage of the clearing process. This in turn would have affected the rest of the data that followed as no two trials were stopped at exactly the same point of clearing by trypsin. Improvement While there is little that can be done with how the human eyes perceives what clear is, it is possible to have a piece of cleared film to act as a reference for the definition of clear. If possible, more sophisticated equipment such as colorimeters and spectrophotometers can be used to pinpoint the exact degree of clearing across all pieces of film. Comment on Order of Significance This limitation is highly significant because it made it impossible to gauge the clearness for each trial, and hence, the ability to stop the timer at the appropriate time for all pieces of film. In essence, all 25 film strips were stopped at different stages, which was against the intentions of the lab. Because of this, all subsequent data (especially the conclusion) may have been subject to a wide range of inaccuracies. This limitation was also important since it involved different definitions for clear. Ultimately, the presence of this limitation also severely affected the outcome of the trials.

## Different individuals stopped the timer

In addition to not knowing when to stop the timer, different people within a group were also in charge of deciding when the film had cleared. Because of different perceptions of what film should look like once cleared, the degree of clearing was once again affected. Hence, the durations of clear and rate were also rendered inaccurate.

In addition to following the improvements for the limitation where clear is not defined, one person should be in charge of stopping timers, to reduce the effects of different human judgments on what clear meant.

Page 13 of 17

Eugene Hui 12.5 Limitation Number of test tubes in water bath changed Effect of Limitation Throughout each trial, the number of test tubes in a water bath changed constantly, which caused fluctuations in the water temperature. This would have worked against the labs assumption that temperature must remain uniform throughout each trial. At the end, changes in temperature could also have been responsible for inaccurate data. Referring to the data table, it is clear that there were slight differences between the ideal Temperature (ie. hot plate temperature) and the Actual Temperature (ie. water bath temperature). In this respect, all data collected actually pertains to rates and durations in seconds for actual temperatures, and not the ideal temperatures that were provided in the task sheet. Thus, data is also slightly misrepresentative of actual conditions of trypsin at the ideal temperatures. Improvement Have more water baths for groups to use, which would prevent groups from having to interfere with each others work. Furthermore, groups should prevent placing their own test tubes into water baths that are currently undergoing other trials for other groups. Comment on Order of Significance As the independent variable in the lab was temperature itself, the fact that the independent variable was not able to be kept consistent affected the data collected throughout the lab. But the importance of this limitation is second to the lack of a clear definition for clear, which undeniably has a more profound effect on the data. While it is clear that differences existed between ideal and actual temperatures, a valid conclusion was still able to be obtained from the resulting data. Of course, data itself was not as accurate as it slightly misrepresents outcomes under ideal conditions. But in the larger scope of the lab, this is a rather minor limitation that can be easily rectified.

## Hot plate temperature different from water bath temperature

The main reason of the discrepancy between the ideal and the actual temperatures was due to too many test tubes being in a water bath at a given time. By restricting the number of test tubes in each water bath, the amount of discrepancy in temperature can be minimized.

Page 14 of 17

Eugene Hui 12.5 Limitation Comment on Order of Significance Each trial was As seen in the data table, the The main reason for As mentioned, this not conducted actual temperatures were this was due to errors limitation caused data at exactly 5 not spaced at 5 degrees and fluctuations in points to be more cluttered degree apart. Thus, this would have water bath when graphing. However, increments caused some trials to result temperatures. Once the general trend was still in data points to be closer to again, having the same established based on the each other, such as in the number of test tubes in existing data points, which case of the 55 and 60 degree all water baths at a was corroborated by other trials, where actual given time should existing experiments. temperatures were only 3 ensure that all water Improving this limitation degrees apart. At the same bath temperatures are would yield more accurate time, this also causes data indeed spaced 5 results, but its presence points to appear more degrees apart from currently is not a large random. one another. detriment to the lab either. 65 degree hot For the 65 degree hot plate This was a random This limitation could be an plate (where water temperature event that was out of explanation as to why malfunctioned was 63 degrees at the start), the groups control. uncertainty was the highest for one minute the hot plate malfunctioned Simply repeating this for the 65 degree trials. and never rose for a little over a minute, but trial would yield much However, this limitation above 60 temperatures in the water more reliable data of was also restricted to the 65 degrees bath never exceeded 60 rate and duration degree trials alone, but had degrees. The effect of this when temperature is no significant impact on the would be a lower rate and a supposed to be at 65 final outcome of the lab. longer duration for the 65 degrees. Even with this limitation in degree trials. This could also place, a conclusion was still explain why 65 degrees able to be derived. produced data with the greatest standard deviation of 14.4%. Measurement It could have been possible This limitation can be Potentially, this factor could errors that each test tube had improved upon by have significant effects on slightly more or less than the having the same rate itself. However, it is 5 ml of trypsin solution. As person collect the unlikely to have played a the volumes were small, the measurements using a large role in this times lab, margin for error was also test tubes with the given the presence of other quite large. In this sense, least amount of major factors such as more or less trypsin solution uncertainty, thus clear not being defined, would affect the number of reducing unnecessary and fluctuations in water enzymes contained within, human error. At this bath temperatures. ultimately affecting rate. time, it is also important to collect measurements by looking at the meniscus. Effect of Limitation Improvement

Page 15 of 17

Eugene Hui 12.5 Limitation Not enough trials at low and high temperatures Effect of Limitation In this lab, the only trials that explored denaturation were at temperatures of 55, 60, and 65 degrees. However, this was not sufficient in gaining a full understanding of denaturation, especially how a high enough temperature will ultimately cause little to no color change in the film negatives. Furthermore, it was hard to observe how a ten degree increase in temperature caused a twofold increase in rate, since the pattern could only be seen when temperature increased from 40 to 50 degrees (in which the 45 degree rate had some discrepancies as well). Given that the trypsin used was not from humans, the optimum temperature in this lab would therefore not be at 37 degrees. However, since the source of trypsin was not known (different organisms have different optimum temperatures), it was impossible to confirm if the optimum temperature stated in the conclusion was correct. Improvement This can be done by adding more trials at 30, 35, 70, and 75 degrees. While this will certainly take more time, but the data that comes thereafter is very useful in understanding the role of temperature on enzymes. Comment on Order of Significance The lack of trials did not hamper efforts at reaching a conclusion in this lab, but does contribute to a lack of a holistic understanding of temperature and enzyme rates. Following this improvement only adds to a better understanding of enzymes, but the conclusion itself still remains valid.

## Experiment may not have been conducted at ideal pH

For trypsin, the ideal pH is at around 7.5-8.5. (Trypsin, n.d.). If the lab was not conducted within this range, then rates in figure 2 would actually have been slightly slower than what should happened in reality at the given temperatures.

Some labs (please see Existing Experiments section) have utilized human trypsin, which makes it easier to compare to existing literature values. Thus, human trypsin (or any other type of trypsin with existing literature values) should be used. By ensuring that the ideal pH is reached before starting the lab, such as using pH indicators to check the pH of the trypsin solution, and making adjustments as necessary.

This limitation mainly pertains to verifying the findings of the lab, but as little to nothing to do with the outcome of the lab itself. Even if the lab is repeated again, this limitation alone is not enough to have a significant impact on the lab itself.

The lab itself looks at how temperature affects trypsin activity, but does not seek to explore the optimum temperature within trypsin in general. Thus, pH is merely an improvement to achieve maximum accuracy in results, but has little weight on the conclusion derived using this set of data.

Page 16 of 17

## Eugene Hui 12.5

Bibliography: Denatured Enzyme. (n.d.). Science Learning Hub RSS. Retrieved March 11, 2014, from http://www.sciencelearn.org.nz/Contexts/Digestion-Chemistry/SciMedia/Images/Denatured-enzyme Enzymes and Inhibitors. (2010, April 14). Biochemaddict21. Retrieved March 5, 2014, from http://biochemaddict21.wordpress.com/2013/04/14/enzymes-andinhibitors/ Effect of Temperature on Trypsin Activity. (n.d.). Welsh Government. Retrieved March 5, 2014, from https://hwb.wales.gov.uk/cms/hwbcontent/Shared%20Documents/VTC/Phase1/ wales/aslevel/biology/molecules/enzymes/Factorsaffectingenzymefunctioning/E xperimenttheeffectoftemperatureontrypsinactivity/default.htm Gelatin. (n.d.). WebMD. Retrieved March 5, 2014, from http://www.webmd.com/vitamins-supplements/ingredientmono-1051GELATIN.aspx?activeIngredientId=1051&activeIngredientName=GELATIN Trypsin. (n.d.). WebMD. Retrieved March 5, 2014, from http://www.webmd.com/vitamins-supplements/ingredientmono-879TRYPSIN.aspx?activeIngredientId=879&activeIngredientName=TRYPSIN Trypsin. (n.d.). Worthington Biochemical Corporation. Retrieved March 5, 2014, from http://www.worthington-biochem.com/TRY/ Standard Deviation. (n.d.). Easy Calculation. Retrieved March 5, 2013, from http://easycalculation.com/statistics/standard-deviation.php

Page 17 of 17