Lab: DENATURING PROTEINS

Proteins and Denaturing Agents Introduction Proteins are polymers of amino acids. A typical protein may be composed of hundreds of amino acids. The Rgroups of the amino acid may be nonpolar, polar, positively charged, or negatively charged. The primary structure of a protein is the sequence of amino acids, and the secondary and tertiary structures of proteins define the proteins folded state. 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, ionic interactions, disulfide bridges, and hydrophobic interactions.

Changing the conformation of a protein either temporarily or permanently by disrupting these forces is called denaturation. Denaturation results in a loss of protein 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 the formation of a solid in the solution. Reagents or conditions that can cause denaturation are called denaturing agents; these include heat, pH changes, alcohol, and heavy metal salts.

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. The R-groups in the amino acid chain are often charged and can form ionic bonds with a group of opposite charge. Extremes of pH can change the charges on these positive and negative groups, disrupting ionic bonds.

Some reagents, such as ethanol, are capable of forming hydrogen bonds with protein molecules which will disrupt the 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 of the protein. 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.

In this exercise, the effect of several denaturing agents on the protein albumin 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).

PROTEIN DENATURATION or WHAT HAPPENS WHEN YOU FRY AN EGG?

In the egg whites the albumin will change from clear to white.  We will explore how the following denature egg albumin as well as milk casein. When egg white is heated above a certain temperature (i.e. 60 °C) bonds in the protein molecules break and reform in different ways. This causes the protein molecules to denature or lose their characteristic three-dimensional structure. As the proteins uncoil, they tend to interact with each other to form a solid mass or coagulum (the process called coagulation). This explains the changes that we observe as we watch an egg being cooked. However, factors other than high temperature may also produce this denaturation phenomenon.

Heat – done by cooking

Acids & bases – can form ions on some side groups of amino acids

Organic compounds – form their own hydrogen bonds with the amino acids

Heavy metals – react with disulfide bonds