last updated July 29, 2002

Enzyme Lab - Ex. 4

General Overview Assignment Analogy
Catalase Amylase Lipase

Bile Salts

 Proteases 
. . Pepsin
. . .Trypsin
pH Iodine Test Benedict's Test Phenol Red
Quiz   Questions

Enzyme - General Information

In laboratory exercise 4 you investigate five enzymes: catalase, amylase, lipase, pepsin, and trypsin.

As an enzyme works it combines with its substrate and converts it to product(s). You will monitor the activity of the enzymes by observing changes in the amounts of substrate and products. 

                    ENZYME

    SUBSTRATE    - - - - - >     PRODUCTS
Much of the laboratory exercise is "cookbook", meaning that you follow specific, and at times relatively involved directions. Then, after the passage of a short time, you observe the results (e.g., a color change, formation of bubbles). As long as you are careful to follow the directions, the results will be as expected.

The challenge is not so much doing the exercises as it is understanding what you are doing, why you are doing it in the specific manner specified, and what the results mean.

Four of the enzymes have special importance in digestion of food by humans.

Although both pepsin and trypsin are proteases, they require quite different conditions of acidity and alkalinity for their action.

The fifth enzyme, catalase, is found in cells of most tissues.

We will use homogenized chicken or beef liver as a source of catalase. The other enzymes were obtained commercially from companies that extract from animal tissues.
Enzyme Substrate Products Test
Catalase H2O2 H2O 
+ 
O2		 Heat 
bubbles
Enzyme Substrate Products Test
Amylase Starch Maltose I2KI starch
Benedicts sugar
Enzyme Substrate Products Test
Lipase Lipid Glycerol 
+ 
Fatty Acid		 Phenol Red 
Acid
Enzyme Substrate Products Test
Pepsin Protein Peptides 
+ 
Amino Acids		 Disappearance 
of egg white
Trypsin Protein Peptides 
+ 
Amino Acids		 Disappearance 
of egg white
 
General Information
The Virtual Enzyme
Enzyme tutorial - animation
Enzyme kinetics -
Enzyme Reaction Tutorials
Energy, enzymes - problem set
Enzyme kinetics
Enzymes and enzyme activity
Factors affecting enzymes
Effect of pH on enzyme activity
pH tutorial
The role of enzymes in biological reactions -
Factors affecting enzymes -
Enzymes -
Animation - simple
Enzyme animation - carboxypeptidase
Animations - links to animations
Animations and links -


Substrate Catalase Products Test
H2O2 - - - - - > H2O + O2 Heat 
bubbles
Catalase

Hydrogen peroxide (H2O2) is a common by-product of metabolic reactions. In high concentration it is toxic; therefore, its accumulation in cells would be harmful. Most tissues, however, contain the enzyme catalase, which catalyzes the breakdown of peroxide to water and oxygen as follows: 

  SUBSTRATE            ENZYME                 PRODUCTS



 2 H2O2              Catalase      --->    2 H2O  + O2  + heat
The reaction is extremely rapid. The action of the enzyme can be demonstrated easily by the evolution of oxygen in the form of gas bubbles when an extract of a tissue containing the enzyme is added to a dilute solution of hydrogen peroxide. We will use homogenized (ground-up) chicken or beef liver as a source of catalase. Catalase
 
 

References:

Substrate Amylase Products Test
Starch - - - - -> Maltose I2KI 
Benedicts

Amylase

Amylase is an enzyme which catalyzes the hydrolysis of the polysaccharide starch to the disaccharide maltose. Salivary amylase is produced by the salivary glands and pancreatic amylase is produced by the pancreas. If amylase is added to a solution of starch, the starch will be digested to form maltose. 
 SUBSTRATE      ENZYME                       PRODUCTS



 starch       Amylase  ---->   maltose + maltose + --- etc. ---
The rate of the reaction is increased if the enzyme and substrate mixture is brought to body temperature (370 C). The progress of the reaction can be visualized by testing the reaction mixture for (1) the disappearance of the substrate (starch) or (2) the appearance of product (maltose). Two simple tests, iodine test for starch and Benedict's test for sugar are used for this purpose.
Substrate Pepsin Products Test
Protein - - - - -> Peptides 
Amino Acids		 Disappearance 
of egg white
Pepsin is a protease that begins digestion of proteins, breaking them into peptides and amino acids. Pepsinogen, is secreted by gastric glands of the stomach into the stomach. There, in the acid environment of the stomach, pepsinogen is converted into pepsin.

Although both pepsin and trypsin are proteases, they require quite different conditions of acidity and alkalinity for their action.

Substrate Trypsin Products Test
Protein - - - - -> Peptides 
Amino Acids		 Disappearance 
of egg white
Trypsin is a protease secreted into the small intestine by the pancreas. As pepsin, trypsin digests proteins into peptides and amino acids and is made and secreted in an inactive form, trypsinogen.

Although both pepsin and trypsin are proteases, they require quite different conditions of acidity and alkalinity for their action.

Substrate Lipase Products Test
Lipid - - - -> Glycerol 
+ 
Fatty Acid		 Phenol Red 
Acid
Lipase is a fat-digesting enzyme, catalyzing the hydrolysis of fat to fatty acids and glycerol. The main source of lipase is the pancreas. 
SUBSTRATE     ENZYME                     PRODUCTS



 lipid       Lipase ---> fatty acid + fatty acid + fatty acid + glycerol
In the following experiment you will use a solution of commercially available pancreatic lipase to study the hydrolysis of milk fat. To follow the reaction, you will make use of the fact that fats are neutral, while fatty acids are acidic. The release of fatty acids from fats by hydrolysis will increase the acidity (lower the pH) of the reaction mixture. This change can be observed by using the indicator dye, phenol red, which is useful for measuring pH values between 6.8 and 8.4.


An Analogy

Suppose you are interested in purchasing a Pizza store and wish to investigate how productive the store is without the present owner knowing because, you fear the owner will raise the price. So, instead of going into the store and watching what happens and asking to examine the books that record expenses and profits, you decide to watch the store from outside.

You observe how often trucks arrive with pizza dough, pizza toppings (cheese, pepporoni, etc.), and other supplies. You also observe how often workers leave the store to deliver pizzas to customers.

In this analogy, the pizza supplies are the reactants and the boxed pizzas that are delivered to customers are the end products. The workers within the store that shape the dough, add the toppings and place the pizzas in ovens and finally in boxes are the equivalent of the enzymes.

Although we don't actually see the workers doing their job, we can infer that if the store is using large quantities of reactants (dough and toppings) and / or making large numbers of end products (pizzas) that the workers (enzymes) must be very active.

The Assignment

Although you will perform the laboratory exercise working as groups of two or three students, make your report an individual effort.

Complete Tables 4.1, 4.2, 4.3 and 4.4. For each table, record your observations at the time that you conduct the experiment. However, leave the explanations of the results until the end.

Your explanations should tell why what happened did happen, or tell the value of the information observed. If, for example, there was more enzymatic activity in one tube than in another, what was responsible for the difference? What does that observation allow us to learn about enzymes?

Some of the observations that you make are of controls. For these, your explanation might tell what you would not know if the control had not been included. For example, you test solutions labeled maltose and starch with Benedict's solution to learn if sugar is present. You are testing sugar and starch to learn if sugar is present. We expect to find a positive reaction with maltose, indicating that sugar is present. Similarly, we expect to find a negative reaction with starch, indicating that sugar is absent.

But consider the possibilities.

  1. Perhaps the solution was labeled incorrectly and that only distilled water was actually in the bottle labeled maltose
  2. Perhaps both solutions were labeled incorrectly and that the bottle labeled maltose contained starch, while the bottle labeled starch contained maltose
  3. Perhaps the reagent labeled Benedict's solution was made incorrectly and did not work to detect sugar
  4. Perhaps glassware was not cleaned well and was contaminated with sugar.
Thus, the simple control shows that the bottle labeled maltose did contain sugar and also that the Benedict's reagent did react as expected when maltose was present.

As indicated in Table 4, be sure to consider as part of your explanation the pH of the environment in which pepsin and trypsin normally work within the human digestive tract.

pH

pH is defined as the negative log10 of the hydrogen ion concentration. Although this definition is intended to assist the scientist by allowing one to express very small quantities without the use of cumbersome fractions (e.g. 0.000000001 gm / liter), students often have difficulty because of two things.
  1. The more hydrogen ions there are, the smaller the pH number.
  2. A change of each whole number represents a tenfold increase or decrease.
0.00000000000001 gm / l 1 x 10-14 gm / l 10-14 gm / l 14 Basic Least H+
0.0000000000001 gm / l 1 x 10-13 gm / l 10-13 gm / l 13
0.000000000001 gm / l 1 x 10-12 gm / l 10-12 gm / l 12
0.00000000001 gm / l 1 x 10-11 gm / l 10-11 gm / l 11
0.0000000001 gm / l 1 x 10-10 gm / l 10-10 gm / l 10
0.000000001 gm / l 1 x 10-9 gm / l 10-9 gm / l 9
0.00000001 gm / l 1 x 10-8 gm / l 10-8 gm / l 8
0.0000001 gm / l 1 x 10-7 gm / l 10-7 gm / l 7 Neutral
0.000001 gm / l 1 x 10-6 gm / l 10-6 gm / l 6
0.00001 gm / l 1 x 10-5 gm / l 10-5 gm / l 5
0.0001 gm / l 1 x 10-4 gm / l 10-4 gm / l 4
0.001 gm / l 1 x 10-3 gm / l 10-3 gm / l 3
0.01 gm / l 1 x 10-2 gm / l 10-2 gm / l 2
0.1 gm / l 1 x 10-1 gm / l 10-1 gm / l 1 Acidic Most H+
grams [H+] per liter grams per liter grams per liter pH value

Iodine Test for Starch

The starch test consists of adding a drop of I2KI (iodine solution) to the sample to be tested. If the iodine retains its yellow-brown color, starch is absent. If a purple or blue- black color forms, starch is present, and the deeper the color, the greater the amount of starch.
 
No starch Starch
   
No Starch . Starch .
  • test for starch

    •  

    Benedict's Test for Reducing Sugar

    The sugar test is performed by adding a small amount of Benedict's Reagent to the sample and bringing the mixture to a boil. If the Benedict's Reagent retains its clear blue color and no precipitate is formed, sugar is absent. The presence of sugar is indicated by the development of a precipitate which may range in color (indicating increasing amounts of sugar) from green, through yellow and orange, to red. This test depends upon the ability of certain sugars to reduce the cupric (Cu++) copper present in the Benedict's Reagent to the cuprous (Cu+) form.