You are on page 1of 6

The Effect of pH on the Reaction between Pepsin and Egg White

Introduction An enzyme is a biological catalyst (Purchon 2012)[4]. Its most elementary function is to speed up the rate of reaction (Enzymes (n.d.))[5]. Without the existence of enzymes within living organisms, the process of digestion would take weeks and the function of our muscles; nerves and bones will decrease in efficiency (Purchon 2012)[4]. Therefore the activities in living systems are controlled through and dependent on enzymes. Similar to other catalysts, enzymes can be reused multiple times, however their natural properties are easily taken away or altered by heat (Purchon 2012)[4]. In order for enzymes to maintain their natural qualities, they must exist in body temperature and a specific pH (Purchon 2012)[4]. The reason why enzymes are so sensitive to heat and pH is because they are protein molecules (Purchon 2012)[4]. An enzyme will only function within its specific chemical reaction. How enzymes work is that they bind themselves to one reactant also known as a substrate (Enzymes (n.d.))[5], by doing this they are lowering the activation energy of the reaction that they are catalyzing, hence the speed of reaction is increased. There are many different types of enzymes within living organisms catalyzing specific chemical reactions. In this particular experiment, the enzyme pepsin was used. First recognized in 1836, pepsin is a digestive enzyme found in the gastric juices of the stomach of living organisms, mainly mammals (Pepsin 2012)[6]. It breaks down protein in foods such as meat, eggs, seeds and dairy products into peptides. Pepsin is formed by the release of pepsinogen in the stomach (Pepsin 2012)[6]. This is sparked by impulses from nerves and hormonal secretions of gastrin and secretin. The pepsinogen then is exposed to and mixes with the hydrochloric acid and rapidly unfolds and breaks, converting into pepsin (Pepsin 2012)[6]. To break proteins into basic amino acid molecules, the bonds that join the amino acids called the peptide bonds must be broken (Hendrickson 2010)[2]. The reaction that breaks these bonds is called hydrolysis, as a water molecule is required in order for the peptide bonds to split (Hendrickson 2011)[2]. During this reaction the peptide bond breaks, simultaneously the water molecule splits and a new bond is formed with one of the amino acids and the hydrogen atom from water, the other amino acid forms a bond with the other hydrogen atom and an oxygen atom present (Hendrickson 2011)[3]. Pepsin speeds up the hydrolysis of protein and makes this reaction more effective (Hendrickson 2011)[3]. Pepsin performs most proficiently at pH of 1.5 - 2.5, which is the normal acidity of gastric juices (Pepsin 2012)[6]. A pH any higher and the enzyme will cease to function properly (Hendrickson 2010)[2]. The enzyme is important for the absorption of nutrition from the protein consumed. The absence of pepsin would cause the digestive tract to move the food eaten through the stomach and intestine very quickly and would not give the body enough time to absorb the nutrition, as the rate of the hydrolysis reaction is too slow without pepsin (Hendrickson 2010)[3]. The small intestine may have absorbed some partially undigested (Carter 2012)[1] or larger fragments of protein and so from the stomach, small amounts of pepsin are passed into the bloodstream where it breaks it all down. The aim of this experiment is to investigate the effect of different pH levels on the activity of pepsin on proteins. That protein that we used was egg white. The main objective was to model the activity of pepsin in the stomach by showing its actions on protein by simulating the effect with pepsin and egg whites. The experiment was carried out at a number of pH levels to show that pepsin (and all other enzymes) have an optimal pH range in which they perform most effectively, in order to model the effect the changing pH has on the activity of the enzyme.

Enzyme has an optimal pH range and anything outside this range inhibits its activity (Hendrickson 2010)[2], therefore it was hypothesised that the lower the pH, the faster the rate of enzyme reaction (pepsin) activity. Pepsin has an optimal range of pH 2 (Pepsin 2012)[6], which is highly acidic and is found in the stomach of the human body where it digests proteins. This means that any pH less acidic or more basic than this level will not allow the reaction to occur, as these are not the optimal conditions for the activity of the enzyme.

Materials and Method 6 x Test tubes 30cm Pepsin 150ml egg white - suspension 1 x Test Tube Rack 1 x Stop Watch 10ml measuring cylinder 500ml beaker 25ml 1M HCl (pH 0) 25ml Citric buffer (pH 3) 25ml Citric buffer (pH 5) 25ml Phosphate buffer (pH 8) 25ml Phosphate buffer (pH 12) Graduated pipette Universal indicator strips

Method: Six test tubes were collected, labeled one to six and placed on a test tube rack. In each of the labeled test tubes 5ml of egg - white suspension was placed into it using a clean graduated pipette. Test labeled one had 5ml of HCl (pH 0) added into it using a clean graduated pipette. 5mL of Citric buffer (pH 3) was added to test tube 2 and similarly test tube had 5ml of citric buffer (pH 5) added into it using clean graduated pipettes. 5ml of phosphate buffer (pH 8) and 5ml of phosphate buffer (pH 12) was added into test tubes 4 and 5 respectively, using clean graduated pipettes. Test tube 6 was used as the control. The precise pH of each solution was compared by adding two drops of the sample, with a clean pipette, onto a universal indicator. This was done for each sample (Carter 2012). One centimeter of pepsin was added into each test tube 1 using a clean graduated pipette and the stopwatch was started simultaneously. The time in which it took the solution in test one to go clear was what was being measured. The results were recorded into the results table and the process of putting one centimeter of pepsin into test tube 1 and timing how long it takes for the solution to clear and recording the figures attained were repeated for test tubes 2-6 in order to ensure reliability of the results. (Steane R. (n.d.)).

Results

Test Tube

Egg white suspension and pepsin plus: 1M Hydrochloric acid

pH Trial 1 0 8.5

Time Taken for Tube to go Clear (min) Trial 2 8.1 Trial 3 8.03 Trial 4 7.9 Trial 5 8.01 Average 8.11

Citric buffer

11.45

5.3

7.1

5.9

5.4

7.03

Citric buffer

No results after 15min No results after 15min No results after 15min No results after 15min

12.6

13.03

11.5

10.98

12.03

Phosphate buffer

No results after 15min No results after 15min No results after 15min

No results after 15min No results after 15min No results after 15min

No results after 15min No results after 15min No results after 15min

No results after 15min No results after 15min No results after 15min

Phosphate buffer

12

Control

14 12 10 8 6 4 2 0

Time (min)

Average Time Taken for Protein to Turn Egg white Suspension at Different pH Levels Clear

5 pH

12

Control

The enzyme seemed to work fastest at a pH of around 3. The second fastest time the enzyme took to render the egg white suspension clear was at pH 0 and any pH at 5 or above took 12min or more as the pH increased. Therefore the enzyme works best at a lower pH, but at an optimal pH of 3. Discussion The results from this experiment support the hypothesized statement. It was hypothesized that the lower the pH, the faster the rate of enzyme reaction (pepsin) activity and this was proven to be true, the average time for pepsin to render the eggwhite suspension clear was lowest at the lower pH of 0 and 3. It took pepsin 12 minutes to turn the egg white suspension clear at pH 5 and over 15 minutes for any pH above 5 such as pH 8 and pH 12. The quickest time it took pepsin to turn to the egg white suspension clear was at pH 3, and this makes sense because it is stated above that pepsin has an optimal range of pH 2 (Pepsin 2012)[6]. Therefore the results obtained suggest that any pH above 3 will inhibit the full function of pepsin and arrests the reaction between egg white and pepsin completely or makes it move very slowly. Therefore, according to the results obtained from this experiment pepsin performs most effectively at pH 3. This makes sense as pepsin performs most proficiently at a pH of 1.5 - 2.5, which is the normal acidity of gastric juices (Pepsin 2012)[6]. Therefore any pH below or above this range will inhibit or slow down the function of pepsin. Hence causing a decrease in the rate of digestion in humans as pepsin is a biological catalyst that breaks down protein into basic amino acids through the hydrolysis reaction (Hendrickson 2010)[2]. These results obtained and observations made were expected and this is seen through the extent as to which it supports the hypothesis. The major sources of possible error were the timing of how long it took for the pepsin to render the egg-white clear as it was only guessed when the egg white was clear and its level of clearness was not consistent with every trial, only similar or close to it. During the experiment there were different people within the group guessing when the egg white had turned clear and in order to improve this error maybe one person should have been nominated to make this judgment so that all the results were consistent throughout the whole experiment. In order to maintain accuracy there were a few things that were controlled such as the amount pepsin, egg white and acid or buffer solution used. The type of instrument to measure all of these solutions, which was a clean graduated pipette. To avoid contamination, a different pipette head was used for each type of solution. The original method was altered slightly and some features were changed due to the type of solutions that were available and some new discoveries that we made during the execution of the experiment. The pH solutions were changed, as the hydrochloric acid pH 2 and sodium chloride pH 9 & 14 solutions were not available. Therefore the solutions available with pHs that were close to the original values were used, such as Citric buffer pH 3&5, and phosphate buffer pH 8&12. We decided that instead of using deionized water for one of the test tubes to make a neutral pH we would swap it with citric buffer with pH 5, to test if this affects pepsin activity. Another major change was the hot water bath. That step was taken out completely as it was discovered during the experiment that after some time the hot water boiled the egg white suspension inside the test tube before any results could be recorded. Therefore the hot water bath was proven to be unsuccessful and that step was taken out. Furthermore, it was originally stated to measure 5cm3 of pepsin, however that proved to be too difficult, hence it was decided that measuring 5ml of pepsin would be a much easier and more accurate option.

There are quite a few improvements that can be made to this experiment if someone were to repeat it. In order to increase the amount of controlled variables, nominate one person to do each task such as measuring out the egg white suspension, acid and or buffer solution, pepsin and placing each into separate test tubes. Also a person to time and observe when the egg white suspension has gone clear so that the judgment remains uniform across all trials. During the process of this experiment, in designing and implementing the experimental protocol, a lot of knowledge was gained about enzymes and their function. There were some difficulties in the sense that communication was found to be quite challenging especially with the age difference within members of the group, its was hard to contact one another, but this was overcome as laboratory time was used effectively as group discussion times. Overall it was a simple and straightforward task. During this experience a lot of valuable insight was learnt about the enzyme pepsin, its purpose, function and the environment as to which it works best in. This was an enjoyable experience and the focus topic was very interesting and engaging.

References 1. Carter, J.S. 2012. Effects of Antacids on Pepsin. Retrieved May 16, 2012 from http://biology.clc.uc.edu/courses/bio115/pepsin%20intro.htm 2. Hendrickson, K. 2010. Pepsin Enzyme Function. Retrieved May 13, 2012, from http://www.livestrong.com/article/313774-pepsin-enzyme-function/ 3. Hendrickson, K. 2011. Action of Pepsin on Proteins. Retrieved May 13, 2012, from http://www.livestrong.com/article/430061-action-of-pepsin-on-proteins/ 4. Purchon, N.D. 2009, Gondor Design Biology. Retrieved May 13, 2012, from http://purchon.com/wordpress/biology/?page_id=98 5. Enzymes (n.d.). Retrieved May 13, 2012, from http://www.elmhurst.edu/~chm/vchembook/570enzymes.html 6. Pepsin 2012. Encyclopdia Britannica Online. Retrieved May 13, 2012, from http://www.britannica.com/EBchecked/topic/450873/pepsin

You might also like