To determine the optimum pH for two different proteolytic enzymes.

Procedure

1. A water bath was set up and the temperature was adjusted to 37℃. The constancy of temperature was checked with a thermometer.
2. 3 ml each of buffer solution of pH 1.0 was pipetted in to three labeled test tubes. This step was repeated for other buffer solutions. Thus, a total of fifteen test tubes (A1, A2, A3 / B1, B2, B3 / C1, C2, C3 / D1, D2, D3 / E1, E2 ,E3) of buffer solutions were prepared.
3. The tubes were divided into three groups (Group1: A1, B1, C1, D1, E1 / Group2: A2, B2, C2, D2, E2 / Group3: A3, B3, C3, D3, E3) and a strip of photographic film was added to each tube. All the tubes were left in the prepared water bath for 10 minutes to reach working temperature.
4. 3 ml each of pepsin was pipetted to the first group (Group1) of test tubes, 3 ml each of trypsin was pipetted to the second group (Group2) and 3 ml each of distilled water was pipetted to the remaining group(Group3).
5. The time taken for the complete disappearance of gelatin on each film was recorded. The tubes were swirled from time to time.
6. The results were tabulated. If the gelatin coats were still remained in the file for over one and a half hours, the time might be considered to be infinity.
7. The experiment was repeated with other buffer solutions of different pH values.

Results

The time of complete disappearance of gelatin on each film was recorded.

Enzymatic Activities ∞  ∞ Rate of reaction

By the graph of rate of enzymatic reaction against pH of buffer solutions, the optimum pH of pepsin is        and the optimum pH of trypsin is        .

Discussion

                                Enzymes are proteins. The functioning of the enzyme is determined by the shape of the protein. The arrangement of molecules on the enzyme produces an area known as the active site within which the specific substrate(s) will "fit". It recognizes, confines and orients the substrate in a particular direction. The use of enzymes can lower the activation energy of a reaction.

At boiling temperature, enzymes are denatured. Enzyme is reusable; it is not consumed in a reaction. As substrates are used up during a reaction, the chance of their colliding with enzyme molecules is decreasing, this results in a fall in reaction rate. The optimum temperature for most enzyme, including human enzymes, lies between 45-50oC (NOT 37oC). Soaking the clothes in water at 37oC can shorten the soaking time because the enzyme action is faster at a higher temperature. (NOT because enzyme works best at 37oC).

Conclusion

Enzymes allow many chemical reactions to occur within the homeostasis constraints of a living system. Enzymes function as organic catalysts. A catalyst is a chemical involved in, but not changed by, a chemical reaction. Many enzymes function by lowering the activation energy of reactions. By bringing the reactants closer together, chemical bonds may be weakened and reactions will proceed faster than without the catalyst. Enzymes can act rapidly, as in the case of carbonic anhydrase (enzymes typically end in the -ase suffix), which causes the chemicals to react 107 times faster than without the enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells to the blood. There are over 2000 known enzymes, each of which is involved with one specific chemical reaction. Enzymes are substrate specific. The enzyme peptidase (which breaks peptide bonds in proteins) will not work on starch (which is broken down by human-produced amylase in the mouth). Enzymatic pathways form as a result of the common occurrence of a series of dependent chemical reactions. In one example, the end product depends on the successful completion of five reactions, each mediated by a specific enzyme. The enzymes in a series can be located adjacent to each other (in an organelle or in the membrane of an organelle), thus speeding the reaction process. Also, intermediate products tend not to accumulate, making the process more efficient. By removing intermediates (and by inference end products) from the reactive pathway, equilibrium (the tendency of reactions to reverse when concentrations of the products build up to a certain level) effects are minimized, since equilibrium is not attained, and so the reactions will proceed in the "preferred" direction.