Optimal Conditions For Everyday Enzymes

Procedure

1. 20g of meat tenderizer was mixed with 200mL of distilled water to create the enzyme solution.
2. The 200mL of solution was divided into seven 50mL beakers, with 20mL of solution in each.  The beakers were labeled 1-7, beaker 4 being the control.
3. The pH of beaker 4 was tested with the pH meter, by removing the cap and placing it in the solution.  The pH was found to be 7.1, meaning that the enzyme solution before adding NaOH or HCl was approximately neutral.  This original pH acted as a starting point for beakers 1-3, and 5-7, and was used to calculate how much NaOH or HCl must be added to reach desired levels of pH for the controlled environments.  (The pH of beaker 4 was recorded in Observation Chart 2 and 3)
4. 10mL of HCl (0.1M) was measured in a graduated cylinder, and then poured into a beaker.  It was tested for pH using an electronic pH meter, which had a reading of 1.03.  (This information was recorded in Observation Chart 1)
5. 10mL of NaOH (1.0M) was measure in a graduated cylinder, and then poured into a beaker where its pH was tested.  The pH meter gave the NaOH a reading of 13.4.  (This information was recorded in Observation Chart 1)
6. NaOH had a concentration ten times that of the HCl, so the NaOH was diluted to the same concentration as the HCl.  108mL of distilled water was added to 12mL of NaOH 1.0M in a 250mL beaker, to produce 120mL of NaOH 0.1M (calculations in analysis section).  The diluted NaOH had a pH reading of 12.92. (This information was recorded in Observation Chart 1)

7. 300mL of distilled water was poured into a 500mL beaker, and tested for pH.  Its reading was 6.7 (This information was recorded in Observation Chart 1).  This water also served as the rinsing agent for the pH meter in between readings.
8. Due to the large amount of acid and base that were needed to change the levels of pH for samples 1 and 7 to the desired level, the samples were transferred into 250mL beakers, and labeled accordingly.
9. 77mL of HCl was added to sample 1 (calculations in analysis section), and it was tested with the pH meter, and had a reading of 1.7.  (The information was recorded in Observation Chart 2 and 3)
10. Step 9 was repeated with samples 2 and 3, with reducing amounts of HCl to achieve two environments with a lower acidity, each one closer to a pH of 7.  (The information was recorded in Observation Chart 2 and 3)
11. 77mL of NaOH was added to sample 7 to raise the pH (calculations in analysis section), and it was tested with the pH meter, and had a reading of 12.81.  (The information was recorded in Observation Chart 2 and 3)
12. Step 11 was repeated with samples 5 and 6, with reducing amounts of NaOH to achieve two environments with decreasing pH, each one getting closer to 7.  (The information was recorded in Observation Chart 2 and 3)
13. Seven identical circular pieces of gelatin were cut out of a Petri dish with the end of a moist test tube.  Each one was placed in a large test tube (labeled 1 through 7).
14. The solution from beaker 1 was added to test tube 1 (containing the gelatin), until it was filled approximately half way.  A stopwatch was started immediately.

15. After 5 minutes, the watch was stopped, and the contents of test tube one was filtered through a tissue and a funnel into a second test tube, labeled 1 b.  (The appearance was recorded in Observation Chart 3)
16. A small (spectrophotometer) test tube was rinsed and cleaned.  It was filled with distilled water and used to calibrate the spectrophotometer.
17. Once the spectrophotometer was calibrated, the small test tube was filled with the contents from test tube 1b.
18. The small test tube was placed in the spectrophotometer, and the reading was recorded in Observation Chart 2)
19. The small test tube was rinsed and cleaned.
20. Steps 14-19 were repeated with samples 2-7.  (Observations were recorded in Observation Charts 2, and 3)

Observations

Observation Chart 1

Observation Chart 2: Quantitative after 5 minute reaction time

Observation Chart 3: Qualitative after 5 minute reaction time

Analysis

Sample Calculations:

How much HCl (0.1M) needs to be added to 20mL of enzyme solution to give it a pH of approximately 1.1?

CHA(VA) +  CHE(VE) =  CHF(VA+VE)

 0.1VA + 7.9E-8(20)  =  7.9E-2(VA+20)

       0.1VA + 1.59E-6 = 1.59 + 7.9E-2VA

               2.06E-2 VA  =  1.59

                            VA  =  77.2mL

Therefore theoretically if 77.2mL of HCl is added to the 20mL enzyme sample, the pH will be 1.1.

How much distilled water and NaOH must be mixed together to give the NaOH a concentration of 0.1M?

     C2V2 = C1V1               

0.1(120) = 1(V1)

         V1 = 12mL

120-12=108

Therefore to reach a concentration of 0.1M of NaOH, 12mL of 1.0M NaOH should be added to 108mL of distilled water.

How much NaOH (0.1M) needs to be added to 20mL of enzyme solution to give it a pH of approximately 12.9?

Since pH + pOH = 14, pOH = 10-1(14-pH)

 COHB(VB) +  COHE(VE) =  COHF(VB+VE)

0.1VB + 10-1(14-7.1)(20)  =  10-1(14-12.9)(VB+20)

           0.1VB + 2.52E-6 = 1.59 + 7.9E-2VB

                   2.06E-2 VB  =  1.59

                                VB  =  77.2mL

Therefore 77.2mL of NaOH needs to be added to the 20mL of enzyme solution to make the pH 12.9.

Conclusion

It was found in this investigation that the meat tenderizer enzyme works best in a slightly basic environment, between a pH level of 8.8 and 10.0.  The highest percent of absorption was 0.126 at a pH of 9.79.  The hypothesis was correct in saying that as the pH increased from very acidic, so would the enzyme function, until a certain point where it would begin to drop again.  What was incorrect about this hypothesis was where the enzyme function would peak.  It was predicted that the enzyme would be most efficient in a neutral environment.  This was not the case.  The enzyme was most efficient in a slightly basic environment.  The percent absorption was relatively low until the environment of the enzyme reached 7.1, and then peaked at a pH of 9.79, and began to become relatively low again after the pH went above 10.55.  These three levels of pH are all slightly basic, showing that the enzyme did work best in a basic environment, opposing the hypothesis.  The hypothesis still has some degree of accuracy, because it was predicted that the percent absorption of the light would gradually go up as the pH rose from very acidic, and that the enzyme function will peak at its optimal pH, and then begin to decline as the pH became higher.  This part of the hypothesis was supported by the observations.

Discussion

        Although this lab had results that were strong enough to draw conclusions from, there is no doubt that there were some sources of error that could be improved upon.  The concentrations of the NaOH and the HCl were not as labeled, which led to many errors in calculation during the lab.  The pH suggested by the labeled concentration is what was used to calculate how much acid or base should be added to the enzyme solution to alter the pH.  This led to the pH of the solutions to be not quite what they were intended to be.  This was accounted for by the use of the electronic pH meter, which allowed the pH of the final solution to be accurately recorded.  Although there was no error in the listing of the pH, if the desired pH was attained, perhaps the range of pH would have been more uniform. Thus making the results easier to draw conclusions from.  In a future lab, the pH of the acid or base would be found with a pH meter, and that pH would be used to calculate how much acid or base to add to change the pH in a more accurate manner.

        5 minutes was used as the time of reaction for the enzymes on the gelatin.  Although the % of light absorbed for each sample was different, there would be a greater difference between them if there was a longer reaction time, since they are all functioning at different rates.  For example if two cars were running side by side, one at 10km/h and one at 20km/h, the longer they run, the further ahead the second car will be.  This could be corrected in a future lab by increasing the time of reaction.

        Gelatin is a substance composed of mainly protein, as is meat.  A major site of protein decomposition is in the small intestine.  This is so that proteins can be broken down into amino acids, or peptide chains small enough to be absorbed by the capillaries is the intestine, into the blood stream.  The environment in the small intestine is basic, this is where an enzyme called Trypsin finishes the job that Pepsin started in the stomach, of breaking down proteins.  Since Trypsin is the enzyme that finished the job and prepares proteins for the blood stream, it must be more efficient than Pepsin.  The purpose of meat tenderizer is to put stress on the bonds between amino acids forming the proteins in meat or gelatin, making it easier to chew, eat, and digest.  This is why it mimics the function of Trypsin, by breaking down proteins best in a basic environment.  It makes sense that since our body’s main enzyme for breaking down proteins works in a basic environment, so should an enzyme made for the same function outside of the body.  The enzyme worked so well in the pH of 9.79 because it is so close to the pH in the small intestine due to the presence of the alkaline glycoproteins and bicarbonate ions.