L5 - Analysis of Proteins

objectives

Understandings:

• Proteins are polymers of 2-amino acids, joined by amide links (also known as peptide bonds).
• Amino acids are amphoteric and can exist as zwitterions, cations and anions.
• Protein structures are diverse and are described at the primary, secondary, tertiary and quaternary levels.
• A protein’s three-dimensional shape determines its role in structural components or in metabolic processes.
• Most enzymes are proteins that act as catalysts by binding specifically to a substrate at the active site.
• As enzyme activity depends on the conformation, it is sensitive to changes in temperature and pH and the presence of heavy metal ions.
• Chromatography separation is based on different physical and chemical principles.

Applications and skills:

• Deduction of the structural formulas of reactants and products in condensation reactions of amino acids, and hydrolysis reactions of peptides.
• Explanation of the solubilities and melting points of amino acids in terms of zwitterions.
• Application of the relationships between charge, pH and isoelectric point for amino acids and proteins.
• Description of the four levels of protein structure, including the origin and types of bonds and interactions involved.
• Deduction and interpretation of graphs of enzyme activity involving changes in substrate concentration, pH and temperature.
• Explanation of the processes of paper chromatography and gel electrophoresis in amino acid and protein separation and identification.

Guidance:

• The names and structural formulas of the amino acids are given in the data booklet in section 33.
• Reference should be made to alpha helix and beta pleated sheet, and to fibrous and globular proteins with examples of each.
• In paper chromatography the use of Rf values and locating agents should be covered.
• In enzyme kinetics Km and Vmax are not required.

Chromatography

• Chromatography refers to a variety of laboratory techniques that involve the separation of a mixture to determine the number of analytes (each substance in the mixture), the relative quantity of analytes and sometimes the identity of the analytes in the mixture.
• Chromatography can also be used to separate and purify the analytes for further analysis.
• For protein analysis, the polypeptide chain would be hydrolysed to reconstitute the amino acids and then the various amino acids would be separated from each other.
• Separation could be based on the polarity of the R groups or by the size of the amino acid.
• This would provide the identification and relative quantity of each amino acid present in the protein.
• There are a variety of chromatographic methods, but the simplest is paper chromatography.
• With this method, the mixture (containing hydrolysed amino acids from the polypeptide chain) is applied as a small spot on the bottom of a piece of chromatography paper.
• The very bottom of the chromatography paper is suspended in a solvent, which then travels up the paper by capillary action.
• If the analyte(s) of the mixture are attracted to the solvent, then they will travel up the paper with the solvent.
• If they are not attracted, they will not travel.
• This results in a separation of the analytes.
• Paper chromatography is often done with ink samples to separate the components of the ink, as shown in the diagram below.

• The results of paper chromatography can show the number of analytes, as determined by the number of spots that the mixture has separated.
• Paper chromatography can also show the relative quantity of the analytes, based on measuring the area of each spot.
• The separation also can be a means of purifying the analytes for further study, as the spots can be cut from the paper and dissolved in a solvent.
• The identification of the analytes can also be determined if paper chromatography has been performed with that substance before.
• The distance that the analyte has travelled, relative to the distance travelled by the solvent (referred to as the solvent front), is used to determine the Rf value, the retention factor.
• Each substance would have a unique retention factor value.
• If paper chromatography has been performed with the same solvent before, the Rf value can be used to identify the substance.

• Separation of the amino acids can be based on the polar-nonpolar interactions of the R groups in the amino acids with the solvent.
• Polar R groups will interact and travel only with polar solvents, such as water or methanol.
• Nonpolar R groups will interact and travel only with nonpolar solvents, such as carbon tetrachloride or hexane.
• There are many R groups that have both polar and nonpolar groups, so paper chromatography can separate the amino acids based on the degree of polarity and interaction with the solvent used.
• With twenty different amino acids that could be present in a polypeptide chain, and many of them having R groups that are similar, sometimes more than one chromatographic process is required to ensure complete separation of all amino acids from one another.
• Two-dimensional paper chromatography uses a sequence of two separations with two different solvents (generally one polar and one nonpolar).
• The mixture is applied to the bottom corner of a large sheet of chromatography paper and placed in the first solvent, just as in traditional paper chromatography.
• The analytes will separate in a single column based on their degree of interaction with the solvent.
• The paper is then rotated 90 degrees to make the row of spots the new origin and then the paper is placed into the second solvent.
• The result is separation by two dimensions, so if two analytes travelled together in the first process, they can be separated in the second process.

• When a polypeptide is analysed by paper chromatography, the amino acids do not exhibit colours, as with ink samples.
• In order to "see" where the amino acids have travelled, another compound, known as a locating agent, is often added.
• A locating agent is a small molecule that binds to the amino acid.
• Following chromatography, the locating agent can be applied to the chromatography paper to reveal the location of where the amino acids have travelled.
• A common locating agent for amino acids is ninhydrin, which reacts with the nitrogen in amino acids, forming a complex that is purple in colour.

Gel Electrophoresis

• Sometimes a sample collected contains a mixture of proteins that require separation.
• For example, if a blood or urine sample is collected from a patient, there are many proteins present that require separation before the individual protein of interest can be identified or studied further.
• Or, when a protein is made up of more than one polypeptide chain, as determined by the quaternary stucture, the polypeptide chains must be separated from each other first before each one can be analysed.
• A common method of separating a mixture of proteins is gel electrophoresis.
• This method involves applying samples of protein mixture to cavities cut into a semi-firm gel and applying an electric current.
• The gel contains pores (holes) that the proteins travel through when the current is applied.
• Since polypeptide chains contain negative ionic charges, the polypeptide will travel at a rate dependent on its size, shape or amount of charge.
• As with chromatography, the proteins are colourless, so a stain must be applied in order to visualise the proteins.

• There are two different methods of gel electrophoresis: native and denaturing.
• In native gel electrophoresis, the protein molecule retains its folded structure, so separation is based on both size and shape.
• Smaller, more compact proteins will travel faster through the gel than larger, bulkier proteins.
• For denaturing gel electrophoresis, the proteins are first denatured with heat or a chemical detergent, losing their shape and returning to a linear polymer.
• The linear polymers can travel through the gel more based on size alone, rather than size and shape, resulting in a better separation than native gel electrophoresis.
• Samples of known polypeptides can also be run at the same time to provide a reference for sizing or identification of the polypeptide sample.
• These reference samples are often referred to as a marker or a 'ladder', since the appearance looks like the rungs of a ladder.
• The ladder would be run in a lane separate from the samples that are being analysed and the polypeptide chains can be determined for size, relative to the sizes of the marker.