The binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. An analogous binding process occurs with serum albumin which binds certain small molecular weight compounds and serves as a carrier molecule for these compounds in blood. In this exercise, students use an electrophoretic assay to examine the binding of various dyes to albumin. The results of this graphic analysis show that the binding of dyes to albumin is saturable, specific, compatible, and dependent on the native structure of the protein. The exercise is designed such that each of the eight groups of students performs a different experiment. Each group then describes their results and conclusion to the entire class. This exercise is a valuable experience in analyzing data and provides a fine introduction to enzyme kinetics. The analysis can be performed during a single 3-hour or two 2-hour laboratory sessions and includes: bovine serum albumin, dilution buffer, bromophenol blue, orange G, ponceau S, sodium dodecyl sulfate, and rabbit hemoglobin.
A. INTERACTION BETWEEN MOLECULES IN BIOLOGICAL SYSTEMS
One of the most important principles of modern biology is that two molecules with complementary surfaces tend to bind or stick together, whereas molecules without such surfaces do not. This principle can be illustrated by considering the specificity of enzyme action. Enzymes accelerate the velocity of virtually all reactions that occur in biological systems, including those involved in breakdown, synthesis and chemical transfers. In so doing, they are responsible for performing essentially all the changes associated with life processes.
The general expression frequently used to describe an enzyme reaction is:
Enzyme (E) + Substrate (S)
Enzyme – Substrate Complex (E-S)
Enzyme + Product (P)
1. The term substrate refers to the compound that is acted upon by the enzyme. In general, enzymes exhibit a high degree of substrate specificity in that they usually catalyze only a single chemical reaction.
2. The enzyme binds to the substrate to form an enzyme-substrate complex. This interaction is responsible for the specificity of enzyme action, since only those compounds that “fit” into the substrate binding site can be acted upon by the enzyme.
3. The enzyme is not destroyed during the reaction but rather is set free after the formation of the end product. Thus, the liberated enzyme is available to combine with more substrate to produce more product.
The binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. Analogous binding processes occur when an antibody binds antigen or when a hormone binds to its receptor. In each case, the molecule that binds to the protein molecule is called a ligand and the region of the protein that associates with the ligand is known as the binding site.
B. CHARACTERISTICS OF LIGAND-PROTEIN INTERACTIONS
Protein Structure – The binding site for a ligand usually consists of a cavity or indentation formed on the protein surface by a specific arrangement of amino acids. Many weak noncovalent bonds are formed between the ligand and the amino acid residues that make up the binding site. The formation of these bonds is required for efficient ligand binding and the structure of the binding site is critical for ligand binding. The native structure of the protein outside of the binding site is also necessary for efficient ligand binding because these regions help to maintain the polypeptide chain in the correct position and give the surface of the molecule the appropriate shape. Consequently, when a protein is denatured by heat treatment or by treatment with certain detergents such as sodium dodecyl sulfate, the polypeptide chain unfolds and ligand binding is abolished.
Ligand Structure and Binding Specificity – The binding of a ligand to a protein is a specific process which is highly dependent on the structure of the ligand. For example, the two major groups of sex steroid hormones are the estrogens and the androgens which are found in females and males, respectively. The structures of the major estrogen (called estradiol) and androgen (testosterone) are shown below. Although the structures of these two hormones are quite similar, their actions are very different. Estradiol promotes feminizing effects such as breast growth in both males and females while testosterone causes masculine reactions including muscu¬lar development and hair growth in both sexes. The differences in the biological responses produced by these hormones is due to estrogen and androgen receptor proteins which are found in tissues that respond to these sex steroids. The estrogen receptor protein is found in tissues that respond to estrogen and this protein binds specifically to estradiol but not to testosterone. By contrast, androgen receptor proteins are found in androgen target tissues and these receptors bind androgens like testosterone but not estrogens. Consequently, the differences in the ligand binding specifications of these receptor proteins is required for differences in male and female phenotypes.
Saturation of Protein by Ligand – The biological response to a steroid hormone is a saturable phenomena. That is, high doses of a steroid hormone produce a maximal response which is not enhanced by additional hormone treatment. Like¬wise, the biological effects produced by essentially all ligand-protein interactions can be saturated with excess ligand. This effect is illustrated in Figure 1 which shows a graph of the amount of protein bound to ligand as a function of ligand concentration. The curve obtained for nearly all protein-ligand pairs is a hyperbola like the one shown in the figure. As noted in the figure, an increase of ligand concentration will at first produce a sharp rise in binding. As the concentration continues to rise, the increase in binding slows and the curve flattens. At maximum binding, the protein is saturated with ligand and additional binding does not occur upon a further increase in ligand concentration. The concentration of ligand required to yield half maximal binding defines the dissociation constant (Kd) which provides an index of the affinity of the protein for the ligand. Thus, a Kd of 0.1M/ L of ligand would indicate that the protein binding site is half saturated with ligand when the ligand concentration is present at that concentration. Such a protein has a very low affinity for it’s ligand. In contrast, a Kd of 104 MIL indicates that the protein has a high affinity since it is half saturated at this low ligand concentration. Most specific protein-ligand interactions in biological systems are characterized by Kd values ranging from 10-5 to 10-12M/L
C. DESCRIPTION OF TELLS LABORATORY EXERCISE
Whole blood, or plasma, clots upon standing and if the clot is removed, the remaining straw-colored fluid is called serum. Serum contains a variety of small molecular weight components as well as hundreds of different serum proteins. The major protein in serum is albumin, which functions as a carrier molecule for the transport of certain small molecular weight compounds in blood. Molecules that bind to serum albumin include bilirubin, fatty acids, hormones, and some synthetic dyes.
In today’s laboratory, you will use an electrophoretic procedure to study the binding interactions that occur between serum albumin and three synthetic dyes. The dyes are called Bromophenol Blue, Ponceau S, and Orange G. The basis for the procedure is the observation that the free dyes not bound to albumin migrate faster than albumin or dyes bound to albumin as shown below. This separation will enable you to distinguish between albumin-bound dye and free dye not associated with the protein. The exercise was designed for eight groups of students and each group will perform a different experiment. At the end of the electrophoretic run, each group will present a five minute oral presentation on the results of their experiment.
Dilution Buffer – The buffer contains 0.05M NaCI, 20% Glycerol, and 10mM Tris
Bovine Serum Albumin
Sodium Dodecyl Sulfate (SDS) – 10%
Proteins and dyes are suspended in dilution buffer and the concentration
of each dye is 0.01M.
Each group should select to perform one of the following experiments.
1. Prepare 1.2% agarose gels as described in the first part of this manual.
2. While the gels are cooling, each group should number four small (0.5m1) tubes 1-4 with a water proof marking pen.
3. Add dilution buffer, bovine serum albumin( BSA), and dyes to the four tubes as indicated below.
Group -1. Albumin Binding is Saturable and Ligand Specific
3 Buffer 20µl 15µ1 10µ1 BSA 0 10µ 10µ Bromophenol Blue
10µ1 Group – 7. Ligand Binding is Reversible and Competable
Tube # Buffer BSA Ponceau S Bromopheno l Blue
4 0 10µ1 20µl 1 20µ1 10µ1 10µ1 0
2 15µl 10µ1 10µ1 5µ1
Group – 2. Albumin Binding is Saturable and Ligand Specific 3 10µ1 10µ1 10µ1 10µ1
4 0 10µ1 10µ1 20µl
Tube # Buffer BSA Ponceau S
1 20µ1 0 10µ1
2 15µ1 10µ1 5µ1
3 10µ1 10µ1 10µ1
4 0 10µ1 20µ1
Group – 3. Albumin Binding is Saturable and Ligand Specific
Tube # Buffer BSA Orange G
1 20µ1 0 10µ1
2 15µ1 10µ1 5µ1
3 10µ1 10µ1 10µ1
4 0 10µ1 20µ1 Group – 8. Ligand Binding is Reversible and Competable
Tube # Buffer BSA Ponceau S Bromophenol Blue
1 20µ1 10µ1 10µ1 0
2 15µ1 10µ1 10µ1 5µ1
3 10µ1 10µ1 10µ1 10µ1
4 0 10µ1 10µ1 20µ1
4. Load 15W of each of your samples (Tubes # 1-4) into four adjacent sample wells. Two groups should share one gel.
5. Electrophorese for 10 minutes at 170 volts, and then turn off and disconnect the power supply.
6. Remove the lid from the electrophoresis unit and note the relative gel position of the albumin-bound and free dyes used in your experiment.
7. Resume electrophoresis until the dyes that are not bound to albumin have migrated to within 1 cm of the positive electrode end of the gel. During this time, prepare an outline of your presentation that will be given at the end of the electrophoretic run. The format below may help in the preparation of the outline.
8. At the end of the electrophoretic run, remove the gels from the unit. At this time, you should measure the distance (in ml) migrated by the albumin bound and free dyes.
The Oral Presentation
The following format can be used as a guide for your oral presentation. The presentation should last no more than five minutes, so you must be orga-nized and concise_
2. Introduction – A brief background of the topic is given in the introduction.
3. Objective – The goal of the experiment should be noted.
4. Results – The data collected is presented in the results section. For this presentation, you may use the actual gel, a diagram of the gel, or a figure or table containing the measurements that were made.
5. Discussion – You should emphasize the importance of the experiment, indicat¬ing the interpretations of the results.
6. Conclusion – The conclusion should contain a restatement of the objective of the experiment and whether or not the objective was accomplished.