Lecture 9Outline of Last Lecture I. Tertiary StructureII. Favorable ResiduesA. Secondary StructureB. Tertiary StructureIII. Protein FoldingA. Protein DenaturingB. Denaturing AgentsOutline of Current Lecture I. Protein PurificationA. Protein Purification MethodsII. Gel Filtration ChromatographyIII. Ion Exchange ChromatographyA. Monitoring ProgressIV. Gel ElectrophoresisA. Visualizing the ProteinsCurrent LectureA protein can be purified, in order to be studied. Purifying will separate out individual enzymes for studying. This can be done by crystallography, gene cloning, through the use of antibodies, and through protein sequencing. One method to study proteins is through chromatography. This involves separating a mixture’s components into two phases: a mobile and stationary phase. This can be done through gel filtration chromatography or ion exchange chromatography. This process is gentle and helps to preserve the protein’s structure. This is important, as chromatography is often used to study its function, which is dictated by structure. Electrophoresis is another method used. This separates a mixture’s components based on their charge and/or size. The size of a protein is usually given as its molecular weight in Daltons. This can also be expressed as a sum of all the amino acid component’s masses. This process can be done by polyacrylamide gel electrophoresis or isoelectric focusing. The downside to this method is that the protein becomes denatured. This method is used when the goal is to evaluate purity. Gel filtration chromatography is sometimes referred to as size exclusion chromatography. The mobile phase of this process contains a buffer, which the proteins are soluble in. A column of beads make up the stationary phase. These beads have holes in them larger than most of the proteins in the solution. Smaller substances flow through the holes in the beads and elute, or exit, slowly. The larger molecules will pass through the column quickly, because they can’t go through the beads. Ion exchange chromatography also contains a buffer in the mobile phase. The stationary phase containsbeads as well. Instead, though, the beads are coated in a positive or negative charge that is covalently attachedBCHM 309 1st Editionto them. These are in the form of chloride or sodium counter ions. A specific type is called anion exchange chromatography. The beads only carry a positive charge. Therefore, negatively charged molecules will bind to the beads, displacing the counter ions. The repelled positively charged substances are able to quickly elute. The substances that are bound to the beads can be eluted through two methods. The first is to add a buffer that contains a gradient of counter ions in increasing concentrations. A change in pH will also cause the same effect. During the course of performing a chromatography, the UV absorbance is monitored. The aromatic amino acids in the proteins will have an absorbance around 280 nanometers. A protein that is also an enzyme can have its enzymatic activity monitored. This will help to show how pure a protein is. Normally the purification process has to be repeated multiple times. Polyacrylamide gel electrophoresis is known as PAGE. Sodium dodecyl sulfate PAGE (SDS-PAGE) is a typeof this method often used. Proteins contain a great diversity in size, shape, and charge. This process coats the protein with a negative charge. This causes the proteins to all behave as negatively charged rods. This type of electrophoresis heats the proteins in a mixture of SDS and mercaptoethanol. The mercaptoethanol breaks disulfide bonds. The SDS binds to the hydrophobic regions, imparting the negative charge. This also completelyunfolds the proteins, allowing them to behave in a more uniform manner. All structure, except for the primary, is lost. All enzymatic activity is also gone. Once this is done, the electrophoresis process continues as the sample is added to a gel. An electrical current applied to the gel causes the proteins to move. As all the proteins are negatively charged, they will migrate downwards to the positive end. Larger proteins will move slower, an end up more towards the top. After the current is applied, a dye can be added to visualize the protein
View Full Document
Unlocking...