Introduction
An enzyme refers to a biological catalyst that mediates reactions. In the absence of enzymes, much energy and time would be required attain physical, as well as chemical conditions necessary for a reaction. An enzyme catalyzes the conversion of one molecule forming another molecule (University of Illinois, 2013). The rate at which enzymes catalyze a given reaction is, usually, influenced by several factors including pH, temperature, and the concentrations of the enzyme and substrate. Most of the factors are required to be at their optimum level for maximum activity of the enzyme to be realized.
Increase or decrease from the optimum lever reduces the activity of the enzyme and continued deviation results in total dysfunction of the enzyme (University of Illinois, 2013). The level of pH has a correlation with the rate of activity of a given enzyme. This experiment aimed to understand how pH influences enzyme kinetics.
Experimental Procedure
Two series of 6 test tubes each labeled with the letter “P” or “T” and even numbers from 2 to 12. The letters were used to indicate the enzyme while numbers represented the pH. The first set was labeled P2, P4, P6, P8, P10 and P12 and the second T2, T4, T6, T8, T10 and T12. Using separate 1 mL-pipettes for each buffer solution, 1 mL of buffer was added into every tube. Using separate 2 mL pipettes for pepsin and trypsin, 2 mL of 0.5% pepsin were added to each tube of the first series and 2 mL of 0.5% trypsin to each tube of the second series. Each tube was vortexed.
Into the 370C, the 12 tubes were placed all at the same time and arranged in order to avoid confusion and error. The solutions were allowed to come to temperature for 5 minutes. A 57 mm strip of exposed 35 mm Plus-X film was cut into 12 narrow strips and inserted into the cleft at the end of each of 12 wooden applicator sticks using forceps. Simultaneously, one of the sticks was placed in each of the 12 test tubes so that the film was partially immersed in the enzyme-buffer solution, and the time recorded.
Each tube was removed from the water bath after every two minutes and examined for signs of breakdown of the gelatin layer. The tubes were returned to the bath after the examination. Time when the immersed portion of a strip cleared was recorded, and the strips were removed as they became clear. The strips were given a maximum of 90 minutes to clear.
Result
The clearance of the strip was checked after every two minutes and the results recorded as shown in Table 1 below.
A graphical representation of the results for the two enzymes is as shown in Figure 1 and 2 below. For the pepsin enzyme, no clearance was noted in the tubes that had a level of pH of 2 and 4. Total clearance was noted in all the other tubes by the end of the 6th minute.
Figure 1: A graph of time taken to clear against pH for pepsin enzyme
For the trypsin enzyme, no clearance was noted in the tubes that had a level of pH of 2 and 4. The tube that had a pH of 6.8 only had partial clearance. Total clearance was noted in all the other tubes by the end of the 6th minute.
Figure 2: A graph of time taken to clear against pH for trypsin enzyme
Conclusion
The optimum pH for the pepsin and trypsin enzymes was determined by checking the clearance of an exposed photographic film that consisted of transparent celluloid backing strip to which silver halide particles were held by a thin gel. Trypsin and pepsin hydrolyzed gelatin when conditions were optimum leaving the strip transparent. From the results, the optimum pH for the pepsin is 6.8 and may continue function up to pH 11. On the other hand, trypsin has a higher optimal pH of about 8 since it took more time to hydrolyze the gelatin at pH 6.8.
The optimum pH of pepsin obtained differs for the value in the literature where pepsin is stated to have an optimal pH of 2 (Schmidt-Nielsen, 1997). The optimal pH of trypsin obtains agrees with the optimal pH cited in the literature (Schmidt-Nielsen, 1997). This experiment, therefore, was successful in demonstrating how pH influences enzyme kinetics.
Reference List
Illinois, U. o. (2013). Enzyme Activity and Assays. Retrieved October 2, 2014, from http://www.life.illinois.edu/biochem/455/Lab%20exercises/B-gal/enzymology.pdf
Schmidt-Nielsen, K. (1997). Animal Physiology: Adaptation and Environment. New York: Cambridge University Press.
