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Department of Pure and Applied Chemistry
College of Arts and Sciences
Visayas State University

Chem 31a
Biochemistry Laboratory

Name: Jayvee A. Garcia
Group #: 1
Lab Schedule: M-F 7-10:20
Date Performed: April 30, 2013
Date Submitted: May 10, 2013

Experiment # 7
Enzyme Activity

Objectives: 1. Associate the presence of enzymes with the catalysts of the chemical reaction in living cells. 2. Determine the effect of enzyme concentration, substrate concentration, temperature, pH and heavy-metal salts upon the activity of salivary amylase.
Results:
A. Enzyme Concentration Test tube | Drops of Enzyme | A-1 Starting Time | A-2Time Blue Color Fades | A-3Time for Starch Hydrolysis | 1 | 1 | 9:07 am | 9:42 am | 35 minutes | 2 | 5 | 9:07 am | 9:37 am | 30 minutes | 3 | 10 | 9:08 am | 9:33 am | 25 minutes | 4 | 15 | 9:08 am | 9:28 am | 20 minutes | 5 | 30 | 9:09 am | 9: 24 am | 15 minutes |

Figure 1: Enzyme Activity (Time for Starch Hydrolysis) vs. Enzyme Concentration

B. Effect of pH pH | B-1Starting Time | Time Blue Color Fades | B-2Time for starch Hydrolysis | 3 | 9:17 am | 9:47 am | 30 minutes | 5 | 9:19 am | 9:44 am | 25 minutes | 7 | 9:19 am | 9:49 am | 20 minutes | 9 | 9:20 am | 9:25 am | 5 minutes | 11 | 9: 20 am | 9:25 am | 5 minutes |

Figure 2: Enzyme Activity (Time for Starch Hydrolysis) vs. pH

C. Effect of Temperature Temperature | C-1Starting Time | Time for Blue Color to Fade | C-2Time for starch Hydrolysis | 0⁰C | 9: 25 am | 10: 05 am | 30 minutes | 25⁰C | 9:26 am | 9: 26 am | 0 minute | 37⁰C | 9:28 am | 9:48 am | 20 minutes | 70⁰C | 9:35 am | 10: 00 am | 25 minutes | 100⁰C | 9:36 am | 9:36 am | 0 minute |

Figure 3: Enzyme Activity (Time for Starch Hydrolysis) vs. Temperature

D. Inhibition of Enzyme Activity Compound | D-1Starting Time | D-2Time for Blue Color to Fade | D-3Time for Starch Hydrolysis | AgNO3 | 9: 10 am | 9:48 am | 38 minutes | NaCl | 9:11 am | 9:25 am | 14 minutes | Ethanol | 9: 12 am | 9:29 am | 17 minutes | Water | 9:15 am | 9:24 am | 9 minutes |

Questions: 1. (A) How does the activity of the enzyme change as its concentration is increased?

As the more of the enzyme is added, the time in minutes for the reaction to happen decreases. This means that enzyme activity is increased as the concentration of the enzyme is increased.

What are some reasons for these changes in activity?

The enzyme binds to or somehow interacts with the substrate to speed up the reaction. There are certain sites on the substrate that the enzyme will act on. So as you increase the concentration of enzymes it increases the number of successful collisions and so to a point the rate of reaction is directly proportional to enzyme concentration and the higher the concentration of the enzyme the faster the reaction.

2. (B) What is the effect of pH upon the relative enzyme activity?

Based on the graph, as the pH was increased, the activity also increased, even when the pH reached a high value. The data is somewhat erroneous since there should be an optimum pH for an enzyme and the graph should look like a mountain, the rate of reaction rising then peaks when reaching the optimum pH then declining beyond that optimum pH. The optimum pH of salivary amylase is 6.8 so the graph should’ve showed the least time in minutes close to pH=7.

What are some reasons for this effect?

pH affects the ionization of the amino acids that make up the active site of an enzyme (active site: where the subtrates are attached to). A change in pH causes a change in the degree of the ionization of the amino acids. The active site might change shape so as not to fit the substrates properly. Hence, less enzyme-substrate complex, enzyme activity decreases.

3. (C) How is the enzyme affected by low temperature? By high temperature?

Low temperatures lessen the activity of the enzyme. Like in pH, there is an optimum temperature; going over that temperature will also lessen enzyme activity. Again, the data is wrong since the optimum temperature for amylase is 37oC.

What are some reasons for these temperature effects?

Enzymes are proteins that catalyze reactions in biological systems. They are typically complex and "flexible". As molecules, their bonds are subject to excitation. At low temperatures, do not flex enough to let in the substrate. Also, the substrates and enzymes do not move around (brownium motion).However, as temperature increases so does the vibration of the molecules and the chance that the enzyme is "denatured" or broken and in need of repair. So reaction rate increases with temperature until a maximum is met. After the optimum temperature is reached, the denaturing effect of temperature is greater than its effect on reaction rate. This is why the graph shows a gradual increase until the optimum where it suddenly drops off.

According to your experiment, what is the optimum temperature for the salivary amylase?

According to the experiment, the optimum temperature for salivary amylase is about 25oC

4. (D) Which of the compounds added to the reaction tubes are inhibitors?
Ethanol and AgNO3

Why do these compounds act as inhibitors of enzyme activity? Enzymes are proteins. Proteins can be easily denatured by heavy metals and alcohols, and this denaturation causes changes in protein properties and can lessen protein activity. NaCl is not an inhibitor. In fact, human salivary α-amylase is often activated by NaCl.

Why is an alcohol swab used prior to giving an injection to a patient?
Alcohols can kill microscopic and harmful organisms in the skin around the entry point of the needle. Along with its antiseptic uses, the alcohol also causes the blood vessels to rise to the skin 's surface, making it easier to insert the needle.

Conclusion: Enzymes are proteins found in the body that catalyze reactions in the body. It lowers the activation energy of reaction and thus results to faster rate of reaction. Several factors affect the activity of these enzymes and they include enzyme concentration, substrate concentration, temperature, pH and heavy-metal salts. The activity of the enzyme is directly proportional to the enzyme concentration. Enzymes have optimum pH and temperature and below and beyond these optimum conditions, enzyme activity declines. There are also compounds that inhibit the enzymes like alcohol and heavy metals.

References: http://answers.yahoo.com/question/index?qid=20091102162158AAh8bMx http://www.worthington-biochem.com/introbiochem/enzymeconc.html http://www.worthington-biochem.com/introbiochem/effectsph.html http://jdr.sagepub.com/content/52/5/1148.extract

References: http://answers.yahoo.com/question/index?qid=20091102162158AAh8bMx http://www.worthington-biochem.com/introbiochem/enzymeconc.html http://www.worthington-biochem.com/introbiochem/effectsph.html http://jdr.sagepub.com/content/52/5/1148.extract