Experiment 14: Proteins and Denaturing Agents (adapted with permission of Dr. Ann Willbrand, USC-Aiken) Introduction Proteins are polymers of amino acids. A typical protein may be composed of hundreds of amino acids. The side chains of the amino acid residues may contain nonpolar, neutral polar, acidic, or basic groups. The primary structure of a protein is determined by the sequence of amino acids, but the secondary and tertiary structures of proteins define their natural or native states, which are often folded. This is called the native conformation and is usually the state in which the protein is most active and functional. Proteins are held in their native conformations by a combination of forces: hydrogen bonds, salt bridges (also called ionic interactions), disulfide bridges, and hydrophobic interactions. NH 2 OOC Ar N H S S Ar O C C O H N Hydrophobic hydrogen bonds salt bridge disulfide bridge Interaction Changing the conformation of a protein either temporarily or permanently by disrupting these forces is called denaturation. Denaturation results in a loss of activity. Since the native conformation is usually the most water soluble, disrupting the secondary and tertiary structures causes changes in solubility and frequently results in precipitation of the protein from solution. Reagents or conditions that can cause denaturation are called denaturing agents; these include heat, pH changes, alcohol, heavy metal salts, and anions such as picrate and tannate. Heat can supply kinetic energy to protein molecules, causing their atoms to vibrate more rapidly. This will disrupt relatively weak forces such as hydrogen bonds and hydrophobic interactions. The most common example is observed in cooking an egg. Heat is also used in sterilization to denature and hence destroy the enzymes in bacteria. Extremes of pH can cause a protein to denature. Although the backbone of a protein chain is neutral, the amino acid residues that comprise the protein often contain acidic and basic groups. These groups are usually charged and can form salt bridges with a group of opposite charge. Extremes of pH can change the charges on these acidic and basic groups, disrupting salt bridges. Less drastic changes in pH can also affect the activity and solubility of a protein. Like individual amino acids, proteins have an isoelectric point at which the number of negative charges equals the number of positive charges. This is frequently the point of minimum water solubility. At the isoelectric pH, there is no net charge on the molecule. Individual molecules have a tendency to approach one another, coagulate, and precipitate out of solution. At a pH above or below the isoelectric pH, the molecules have a net negative or positive charge, respectively. Thus when protein molecules approach each other, they have the same overall charge and repulse each other. This prevents coalescence and precipitation. Casein, the major protein in milk, provides a good example. At its isoelectric pH of 4.6 it will precipitate, but it is soluble at the normal pH of milk, 6.6. When milk sours, lactic acid is produced that lowers the pH. Casein precipitates, forming white curds also known as cottage cheese. Reagents such as ethanol that are capable of forming intermolecular hydrogen bonds with protein molecules will disrupt the intramolecular hydrogen bonding within the molecule. A 70% solution of alcohol can be used as a disinfectant, because the alcohol functions to denature the proteins in bacteria. A 70% solution is used because it will effectively penetrate the bacterial cell wall; a 95% solution coagulates proteins at the surface of the cell wall, forming a crust that prevents the alcohol from penetrating into the cell. Salts of metal ions such as mercury(II), lead(II), and silver can form strong bonds with disulfide groups and with the carboxylate ions of the acidic amino acids. Thus, they disrupt both disulfide bridges and salt linkages and cause the protein to precipitate out of solution as an insoluble metal-protein salt. This property makes some of the heavy metal salts suitable for use as topical antiseptics. However, most heavy metal salts are toxic when taken internally, because they precipitate the proteins of all the cells with which they come into contact. Substances high in protein, such as egg whites and milk, are used as antidotes for heavy metal poisoning, because their proteins readily combine with the metal ions to form insoluble solids. The resulting insoluble matter must immediately be removed from the stomach by the use of an emetic to prevent the gastric juices from destroying the protein and once again liberating the poisonous heavy metal ions. Picrate and tannate ions are the conjugate bases of picric and tannic acids, respectively. These negatively charged ions combine with the basic amino acids, which are positively charged. This disrupts intramolecular salt bridges. In the manufacture of leather, tannic acid is used to precipitate the proteins in animal hides. This is commonly known as the tanning process. Tannic acid, which is found in tea, is sometimes used to treat burns. The acid combines with the protein in the exposed areas to form a leathery coating that excludes air and stops the loss of bodily fluids. In this exercise, the effect of several denaturing agents on two different proteins will be studied. Albumin is a simple globular protein. It is soluble in water and dilute salt solutions such as isotonic saline (0.9% NaCl). Casein is the major protein component of milk. Its solubility is very sensitive to pH. Laboratory Activities A. Examine the effect of heat on the solubility of albumin B. Examine the effect of pH changes on the solubility of albumin and casein C. Examine the effects of 95% ethanol, lead(II) nitrate, silver nitrate, and tannic acid on albumin and casein Procedure A. The effect of heat Place about 1 mL of 2% albumin in a test tube and heat it in a hot water bath for a few minutes. Compare the appearance to the albumin solution at room temperature. B. The effect of pH changes Place about 1 mL of 2% albumin in each of four test tubes. Add 3 mL of water to the first. This tube will serve as a control. To the second, add 10% NaOH dropwise until the pH is 14. (To do this, add a couple of drops of NaOH to the tube; stir thoroughly with a stirring rod; then touch the stirring rod to a piece of pH paper to check your pH.) To the third, add 0.5% sodium bicarbonate solution to pH 9, and to the fourth, add 2% HCl to pH 2. Record your observations on the data sheet. Repeat the above tests using 2% casein solution. Record your observations. Compare the results for the two tests. C. The effects of ethanol, lead(II) nitrate, silver nitrate, and tannic acid Place 1 mL portions of 2% albumin in each of 5 test tubes. The first tube is your control. To the second tube, add 1 mL of 95% ethanol; to the third, add several drops of lead(II) nitrate; to the fourth, add several drops of silver nitrate; and to the last, add several drops of tannic acid. Compare each of the test solutions to the control. Record your observations. Repeat the above tests, substituting 2% casein solution for the albumin. Record your observations. Are your results consistent with the results from albumin? Experiment 14 Name:______________________________ Laboratory Record Date:_______________________________ A. Heat effect Record your observations: B. pH Effects Record your observations in the chart: Medium (pH) Effect on Albumin Effect on Casein Water (pH 7) (control) 10 % NaOH (pH 14) 5 % NaHCO3 (pH 9) 2% HCl (pH 2) C. Effects of ethanol, lead(II) nitrate, silver nitrate, and tannic acid Record your observations in the chart: Agent Effect on Albumin Effect on Casein None (Control) 95 % ethanol Pb2+ Ag+ Tannic acid Questions: 1. In your own words, explain the effect of heat on the solubility of albumin. 2. Is casein more soluble in base or in acid? Why? 3. When milk sours, lactic acid is produced and a white precipitate forms. What is this precipitate? Explain what is happening. 4. Did you observe any differences in the solubilities of casein and albumin at pH 2? at pH 9? at pH 14? From your observations, which is more affected by pH, casein or albumin? 4. What kinds of disruptions to the native conformation occur in each of the following? In other words, are hydrogen bonds, disulfide linkages, etc. disrupted? Albumin is heated. Alcohol is added to casein. Metal ions are added to casein. Tannic acid is added to albumin.