Enzymes – Proteins & Amino Acids

Enzymes are proteins produced by all living organisms. These proteins consist of amino acids but what makes them different is how they behave in our body. (Felice) Enzymes are catalysts that make biochemical reactions take place within our bodies take place quickly and efficiently. These reactions would otherwise happen very slowly and or not occur at all. (SYMBIOSIS p59)
These enzymes are highly specific in their functionality and work in different ways. They are the basic elements that activate all functions in the body. They facilitate reactions that build compounds from raw materials within the body, transport elements throughout the body, break down substances and eliminate the waste and unwanted chemicals within our bodies. Simply, enzymes carry out all the body’s biochemical functions. (Felice)

Since enzymes are proteins, each consists of a specific sequence of amino acids. (WORTHINGTON) Weak hydrogen bonds that form between some of the amino acids help to determine the shape of the enzyme. It is this shape that allows the enzyme to fit onto a specific substrate molecule. Figuratively speaking, the enzyme and substrate molecule must fit together like a lock and key. (SYMBIOSIS p60)
The material with which catalysts react is the enzyme’s substrate, which fits into a region of the enzyme called the active site. These enzymes are specific due to their active site. This active site will bind with the substrate to form the enzyme-substrate complex. (BIOLOGY p84) This is where the catalysis takes place. When the catalyst is complete the complex dissociates into enzyme and products. Enzymes lower the energy of activation needed for reactions to take place, they accelerate the rate of reactions. However, they do not determine the direction in which a reaction will go. (SYMBIOSIS p59)

Enzyme activity is influenced by many factors such as varying environmental conditions, such as pH or temperature. Enzymes have a unique three-dimensional shape which determines which reaction the enzyme catalyzes. (BIOLOGY p84) Changes in the body’s temperature or the pH level may alter the three-dimensional shape of the enzyme or alter its rate of activity. When the shape of an enzyme is changed, this process is called denaturing.

An enzyme is inactivated by a change in its shape, which can be altered by anything that disrupts the pattern of hydrogen bonding. Many enzymes function best within a narrow range of temperature or pH level. Substantial changes in either can disrupt their hydrogen bonds and alter their shape. It is the unique bonding pattern that determines the sensitivity of each enzyme to changes in temperature and pH. (SYMBIOSIS p61)

For the purpose of this lab, we will perform three separate experiments to determine how certain enzymes are affected by changes in temperature and pH.

Experiment I: The Influence of Enzyme Concentration on the Rate of Starch Digestion
Amylase is an enzyme found in the saliva of many animals, including humans. Amylase utilizes starch as a source of food. Amylase is responsible for the preliminary digestion of starch by breaking up the chains of glucose molecules in starch into maltose, which is a two-glucose-unit compound. (SYMBIOSIS p62)

Materials and Methods
In this experiment, we will investigate the influence of enzyme concentration on the activity of the enzyme amylase. To help us follow the digestion of starch into maltose by salivary amylase, we will take advantage that starch, but not maltose, turns a dark purple color when treated with a solution of I₂KI, which is normally yellow-amber in color. (SYMBIOSIS p62)
We will vary the concentration of the enzyme amylase to determine that affect, if any, the variation will have on the rate of the reaction. The rate of disappearance of starch in these different concentrations allows a quantitative measurement of reaction rate. (SYMBIOSIS p62)

We will begin the experiment by preparing the Amylase dilution. We will number five test tubes. Using the 5ml graduated pipette, we will add 5ml of distilled water to each tube. Using the graduated cylinders, we will then make serial dilutions. To do this we will add 5ml Amylase to tube #1 and mix by rolling the tube between our hands. In tube #2, we will add 5ml of Amylase from tube#1 and mix; then in tube #3, we will add 5ml of Amylase from tube#2 and mix; then in tube #4, we will add 5ml of Amylase from tube#3 and mix; and finally in tube #3, we will add 5ml of Amylase from tube#4 and mix. Then we will rinse the graduated cylinder thoroughly. (SYMBIOSIS p63)

Next, we will prepare the experimental test tubes. We will begin by numbering the test tubes 1-5. Beginning with test tube #5 of the first set, we will transfer 2ml of the dilution into tube 5 of the second set using a 5ml pipette. We will rinse the pipette thoroughly with distilled water and repeat the procedure for tubes 4, 3, 2 and 1. After the transfers have been carried out, the first test tube set will not be used again in the experiment. (SYMBIOSIS p63)

Now, we will add 1 or 2 drops of I₂KI to each compartment of four rows of a spot plate. We will use a separate row for each concentration of Amylase. Using the second set of tubes, we will proceed with the tests beginning with tube 5. Using a clean pipette, one lab partner will add 1ml of the 1% starch solution to tube #5 and mix by rolling the tube between their hands, while the other partner immediately records the time. This time is 0. Then we will remove 21 drop of the mixture with a disposable Pasteur pipette, and add it to a drop of the I₂KI in the first compartment on the test plate. Then we will sample the reaction mixture at 10 second intervals, each time using a new compartment of the test plate. We will continue this process until the blue color is no longer produced and the I₂KI solution remains its original color (yellow-amber) indicating that all of the starch has been digested. We will repeat the same procedure for tubes 4, 3, 2 and 1. We will finish by recording our findings. (SYMBIOSIS p63-64)

Experiment II: The Effect of pH on Catalase Activity
Catalase is an enzyme that speeds the breakdown of hydrogen peroxide to water and oxygen. Cells of almost all living organisms use Catalase to remove Hydrogen Peroxide, which is a toxic byproduct of metabolism. (SYMBIOSIS p65)

In this experiment we will examine the activity of Catalase at three different pH levels. The reaction between Hydrogen peroxide and Catalase products produces tiny bubbles. The more bubbles, the higher the activity.

Materials and Methods
For this experiment, we will add 1ml of Catalase to three test tubes numbered 1-3. Then we will add 2ml of pH2 buffer to test tube #1; 2ml of pH7 buffer to test tube #2; and 2ml of pH10 buffer to test tube #3. After adding the buffers, we will swirl each test tube and then add a drop of soap to each test tube. Then we will add 2ml of Hydrogen peroxide to each tube and wait 20 seconds. After 20 seconds, we will measure the height of the bubble column. (SYMBIOSIS p66)

The research hypothesis is that the pH level of the solutions will affect the height of the bubble column. For the lower pH level, there should be little or no reaction; and as we go higher on the pH scale, the more bubbles we will see. This is due to the enzyme will have been denatured by the lower pH level.

In test tube #1, there is no reaction within the solution.
In test tube #2, the height of the bubbles is 5cm indicating that there is a reaction within the solution.
In test tube #3, the height of the bubbles in 15cm, showing that this solution has the highest reaction within the solution.

Experiment III: The Effect of Temperature on Rennin Activity
Rennin is a protein digesting enzyme found in the lining of the stomachs of young mammals. Newborn babies and infants produce rennin but it is undetectable in adults. The reason is because the purpose of Rennin is to help solidify milk so that it will stay in the stomach and digestive system as a solid long enough to be digested and absorbed. (SYMBIOSIS p68)

Chemical reactions, as explained above, accelerate as temperature rises. This is partly due to increased temperatures speeding up the motion of molecules causing the substrates to collide more frequently with enzyme active sites. A 10⁰ rise in temperature results in a two to three fold increase in the rate of a particular reaction. However, at higher temperatures, the integrity of proteins can be irreversible denatured. The optimum temperature for activity may vary depending on the structure of the enzyme. (SYMBIOSIS p68)

Materials and Methods
For this experiment, we will number three test tubes 1-3 and then measure and mark each tube 2 cm from the bottom of each tube with a wax pencil. To tube #1, we will add 4ml of refrigerated milk; however we will add 4ml of warm milk to tube numbers 2 and 3. Then we will add Rennin in different temperatures to each tube. For test tube #1, we will add 3 drops of refrigerated Rennin; for test tube #2 we will add 3 drops of warmed Rennin; and to test tube #3 we will add 3 drops of boiled Rennin. After adding the Rennin, we will place each tube in different temperatures for a total of 15 minutes. We will place test tube #1 in the refrigerator; test tube numbers 2 and 3 will both be placed in a warm water bath. After the 15 minutes have elapsed we will observe any changes in the solutions. (SYMBIOSIS p68-69)

The research hypothesis is that the Rennin will react by causing the milk solutions to solidify as they would in the stomachs of infants and small mammals. I hypothesize that the refrigerated Rennin and milk solution will have little, if no, reaction; The warmer Rennin and milk solution will be more solidified than the refrigerated solution; and finally I hypothesize that the solution in which the Rennin was boiled and the milk was warmed will have the highest solidity of the three. (SYMBIOSIS p68-69)

In test tube #1, we find the milk solution is barely solidified, almost not affected at all. This is due to the fact that the temperature was significantly lower than that of the other solutions causing little reaction with the Rennin.

In test tube #2, the milk solution is slightly more solidified than the refrigerated solution. This is due to the fact that the temperature of the milk and Rennin was warmer in temperature causing the solution to react by solidifying. However, giving the solution a warmer environment by being placed in a warm water bath also aided in causing the solution to further solidify.

In test tube #3, the milk solution was warm and the Rennin was boiling, therefore causing an almost instant reaction in the solution. However, after being given a warm water bath for 15 minutes it was almost completely solid. Therefore, my hypothesis regarding the affect of temperature on the enzymes is proved correct.

Works Cited

Pearson Custom Publishing. SYMBIOSIS: the Benjamin Cummings custom laboratory program for the biological sciences. Boston, Massachusetts 2008.

Pearson Education, Inc. BIOLOGY: Concepts & Connections. San Francisco, California 2009.

What is an Enzyme. Karen DeFelice. 15 April 2006. http://www.enzymestuff.com/

Introduction to Enzymes. Worthington Biochemical Corporation. February 2009. http://www.worthington-biochem.com/IntroBiochem/default.html