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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...
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