Enzymes are biological catalysts that carry out the thousands of chemical reactions that occur in living cells. They are generally large proteins made up of several hundred amino acids, and often contain a non-proteinaceous group called the prosthetic group that is important in the actual catalysis.
     In an enzyme-catalyzed reaction, the substance to be acted upon, or substrate, binds to the active site of the enzyme. The enzyme and substrate are held together in an enzyme-substrate complex by hydrophobic bonds, hydrogen bonds, and ionic bonds. The enzyme then converts the substrate to the reaction products in a process that often requires several chemical steps, and may involve covalent bonds. Finally, the products are released into solution and the enzyme is ready to form another enzyme-substrate complex. As is true of any catalyst, the enzyme is not used up as it carries out the reaction but is recycled again and again. One enzyme molecule can carry out thousands of reaction cycles every minute.
     Each enzyme is specific for a certain reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The "business" end of the enzyme molecule, the active site, also has a specific shape so that only one or few of the thousands of compounds present in the cell can interact with it. If there is a prosthetic group on the enzyme, it will form part of the active site. Any substance that blocks or changes the shape of the active site will interfere with the activity and efficiency of the enzyme. If these changes are large enough, the enzyme can no longer act at all, and is said to be denatured. There are several factors that are especially important in determining the enzyme's shape, and these are closely regulated both in the living organism and in laboratory experiments to give the optimum or most efficient enzyme activity:

Salt concentration: If the salt concentration is very low or zero, the charged amino acid side-chains of the enzyme will stick together. The enzyme will denature and form an inactive precipitate. If, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked, new interactions occur, and again the enzyme will precipitate. An intermediate salt concentration such as that of blood (0.9%) or cytoplasm is optimum for most enzymes.

pH: The H⁺ concentration of an aqueous solution is known as the pH, and it is measured on a logarithmic scale. The scale runs from 0 to 14, with 0 being the highest in acidity and 14 the lowest. Neutral solutions have a pH of 7. Acid solutions have a pH less than 7; basic solutions have a pH greater than 7. Enzyme amino acid side chains contain groups such as -COOH and -NH₂ that readily gain or lose H⁺ ions. As the pH is lowered, an enzyme will tend to gain H⁺ ions, and eventually enough side chains will be affected so that the enzyme's shape is disrupted. Likewise, as the pH is raised, the enzyme will lose H⁺ ions and eventually lose its active shape. Many enzymes have an optimum in the neutral pH range and are denatured at either extremely high or low pH. Some enzymes, such as those which act in the human stomach where the pH is very low, will have an appropriately low pH optimum. A buffer is a compound that will gain or lose H⁺ ions so that the pH changes very little.

Temperature: All chemical reactions speed up as the temperature is raised. As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme catalyzed reaction is raised still further, an optimum is reached: above this point the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme molecules starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around 40-50C, but some are still active at 70-80C, and a few even withstand being boiled.

Small molecules: Many molecules other than the substrate may interact with an enzyme. If such a molecule increases the rate of the reaction it is an activator, and if it decreased the reaction rate it is an inhibitor. The cell can use these molecules to regulate how fast the enzyme acts. Any substance that tends to unfold the enzyme, such as an organic solvent or detergent, will act as an inhibitor. Some inhibitors act by reducing the disulfide (-S-S-) bridges that stabilize the enzyme's structure. Many inhibitors act by reacting with side chains in or near the active site to change or block it. Others many damage or remove the prosthetic group. Many well-known poisons, such as potassium cyanide and curare, are enzyme inhibitors which interfere with the active site of a critical enzyme.
 

     In this exercise, you will study the enzyme catalase, which accelerates the breakdown of hydrogen peroxide (a common end product of oxidative metabolism) into water and oxygen, according to the summary reaction:

2H₂O₂ + catalase ----> 2H₂O + O₂ + catalase.

This catalase-mediated reaction is extremely important in the cell, because it prevents the accumulation of hydrogen peroxide, a strong oxidizing agent which tends to disrupt the delicate balance of cell chemistry.
     Catalase is found in animal and plant tissues, and is especially abundant in plant storage organs such as potato tubers, corms, and in the fleshy parts of fruits. You will isolate catalase from potato tubers and measure its rate of activity under different conditions. A glass, fiber filter will be immersed in the enzyme solution, then placed in the hydrogen peroxide substrate. The oxygen produced from the subsequent reaction will become trapped in the disc and will give it buoyancy. The time measured from the moment the disc touches the substrate to the time it reaches the surface of the solution is a measure of the rate of the enzyme activity.
 

A. Extraction of Catalase
1. Peel a fresh potato tuber and cut the tissue into small cubes. Weigh out 50 g of tissue.

2. Place the tissue, 50 ml of cold distilled water, and a small amount of crushed ice in a blender.

3. Homogenize for 30 seconds at high speed. From this point on, the enzyme preparation must be carried out in an ice bath.

4. Filter the potato extract, then pour the filtrate into a 100-ml graduated cylinder. Add cold distilled water to bring up the final volume to 100 ml. Mix well. This extract will be arbitrarily labelled 100 units of enzyme per ml (100 units/ml) and will be used in Tests 1 to 5.
 

B. Test 1: Effect of Enzyme Concentration
     Before considering the factors that affect enzyme reactions, it is important to demonstrate that the enzyme assay shows that the enzyme actually follows accepted chemical principles. One way to demonstrate this is by determining the effect of enzyme concentration on the rate of activity, while using a substrate concentration that is in excess.
     Label four small cups as follows: 100, 75, 50, and 0 units/per ml. Prepare 40 ml of enzyme for each of the above concentrations.

enzyme (ml) + cold distilled water (ml) = units/ml

40*                              0                              100
30                              10                                75
20                              20                                50
0                                40                                  0

* save this undiluted enzyme for Tests 2 to 5

Keep your catalase preparations in the ice bath. Label an identical set of cups for the substrate. Into each of these beakers, measure out 40 ml of a 1% hydrogen peroxide solution.
     Using forceps, immerse a 2.1-cm glass, fiber filter disc to one-half its diameter in the catalase solution you have prepared. Allow the disc to absorb the enzyme solution for 5 seconds, remove and drain by touching the edge to a paper towel for 10 seconds. Drop the disc into the first substrate solution. The disc will sink rapidly into the solution. The oxygen produced from the breakdown of the hydrogen peroxide by catalase becomes trapped in the fibers of the disc causing the disc to float to the surface of the solution. The time (t) in seconds, from the second the disc touches the solution to the time it again reaches the surface is an indirect measure of enzyme activity. Record your results in a table in your notebook. After you have collected data from all other groups, calculate means and standard deviations for each concentration, and construct and label a graph in your notebook, and plot your results. What are the independent and dependent variables in this small experiment? How does enzyme activity vary with enzyme concentration?
 

C. Test 2: Effect of Substrate Concentration
     To determine the effect of substrate concentration on enzyme activity obtain eight 50-ml beakers and label them as follows: 0%, 0.3%, 0.7%, 1%, 3% H₂O₂. Add 40 ml of the proper (as outlined above) H₂O₂ solution to each beaker. Make sure that the substrate solutions reach room temperature before beginning your assay. Using the filter disc procedure described above, and the undiluted enzyme from Test 1, determine the rate of the reaction at the various substrate concentrations. Record your results in in a table in your notebook. Plot the class results in a labelled graph in your notebook.
     How is the rate of enzyme activity affected by increasing the concentration of the substrate? What do you think would happen if you increased the substrate concentration to 10% H₂O₂? Does changing the substrate concentration exhibit the same effect as changing the enzyme concentration?
 

D. Test 3: Effect of Enzyme Inhibitor
     Hydroxylamine attaches to the iron atom (a part of the catalase molecule) and thereby interferes with the formation of enzyme-substrate complex. Add 5 drops of 10% hydroxylamine to 1 ml of enzyme extract and let it stand for 1 minute. Prepare a control solution to test. Then measure the activity of each solution. Use 40 ml of 1% H₂O₂ for the substrate. Record data in a table in your notebook. Explain the results.
 

E. Test 4: Effect of Temperature
     Using 40 ml of a 1% hydrogen peroxide solution as the substrate, and 5-ml aliquots of the 100 units/ml enzyme solution, measure the enzyme activity in the usual manner. Run the reactions in the water baths at different temperatures, such as 4, 22 (room temperature), 30, and 37 C. The catalase and substrate should be brought to the testing temperature before they are used. Record the exact temperature and your data in a table in your notebook and plot the class results in a labeled graph. From these data, what can you conclude about how temperature affects enzyme activity? How would you explain the results?

F. Test 5: Effect of pH
     Obtain 5 50-ml beakers, and label them as follows: pH 4, pH 6, pH 7, pH 8, and pH 10. Into each beaker, pour 10 ml of enzyme preparation and 30 ml of buffer solution at the appropriate pH.  Using 40 ml of a 1% hydrogen peroxide solution as the substrate, measure the enzyme activity in the usual manner.
     How does pH affect enzyme activity? Would you expect similar results with salivary amylase, which is an enzyme that breaks down starch? Would you expect similar results with pepsin, which is an enzyme of your stomach?

     In their papers, biologists typically report the results of their studies in either tables or figures. You will need to be able to construct both figures and tables when writing lab reports for this class and for future classes.

A. Tables
     Rarely would a table present the raw data as we have in our small experiments. Tables used for original data are simply tools for you to organize your data as it is collected. In most biological studies, the data sets are so large that you would not be able to present them all when writing a scientific paper. Instead, when writing a paper about an experiment, tables are used to summarize the data, as well as to show the results of any statistical analyses that were conducted.
     In a scientific paper or in a lab report, tables are always numbered consecutively (Table 1, Table 2, Table 3, etc.). Also, there is always a title or caption that informs the reader what results are being presented. The caption is typically placed directly above the table. Units of measurement should always be included, either in the caption or in the column or row headings. In addition, the sample size is typically include, either as a part of the table itself or in the caption.

B. Figures
     A figure is a general term used for graphs, pictures, illustrations, and diagrams. These are also numbered consecutively in a scientific paper or lab report (Figure 1, Figure 2, etc.) and should be accompanied by a figure legend (analogous to a caption or title). In contrast to table titles, the figure legends are typically placed directly below the figure.
     In many experiments, graphing the results is one of the first things a scientist does in order to get a feeling for the general relationship between the independent and dependent variables. It is also a very widely used method of presenting the results to colleagues in presentation, papers, and reports. When constructing a graph, you should follow these guidelines:

1. To plot the values accurately for a lab report, use either graph paper or a graphing program on a computer. For your lab notebooks, you can simply construct the axes and plot the data on whatever type of paper you are using. Whenever you plot a set of data by hand, you should use a ruler to neatly draw the axes..

2. Graph the independent variable on the horizontal axis (x axis) and the dependent variable on the vertical axis (y axis).

3. The numerical range of values on the x or y axis should be appropriate for the data. For example, if the data from an experiment fall between 0 and 10, the range of values plotted should not be 0 to 100. Also, divide the full range of values into appropriate, evenly spaced intervals, such as 0, 1, 2, 3, 4, 5, 6, Ö, not 0, 1, 4, 5, 6, 8,Ö If you choose to use graph paper, you should use most of the graph space.

4. Label each axis with the name of the variable and the unit of measure. If you have more than one set of data on the same axes, you will need to indicate which symbols or shading patterns are associated with which data set.

5. Choose the appropriate kind of graph. The two main kinds of graphs you will see in scientific papers and will have a chance to use this semester are bar graphs and line graphs. Line graphs are best used when the independent variable can take on a continuous set of values (e.g., 0-100). Bar graphs are often more appropriate when the independent variable can take on a limited set of discrete values or categories. In line graphs, each piece of data is plotted as a separate point on the graph. It is sometimes appropriate to draw lines between the points; in other cases, it is not. In bar graphs, a bar is constructed from the appropriate value or category on the x axis up to the correct value of the dependent variable.