topic guide 1.3: separation of proteins

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1: Biochemistry of macromolecules and metabolic pathways

Preparative purification techniques produce a large quantity of purified proteins for use; examples of industrial use include the preparation of enzymes, isolating soy proteins in the nutritional industry and preparation of insulin for medical use. A considerably smaller amount of protein is produced during analytical purification for research into the structure of different proteins. In medical research, analysing the structures of proteins is very important for the diagnosis of disease.

On successful completion of this topic you will: • be able to use biochemical practical skills and cognate techniques (LO4).

To achieve a Pass in this unit you need to show that you can: • plan types of protein separation in terms of the theory and practice

involved (4.1) • carry out a separation and purification of protein from a simple mixture,

using safe practices (4.2) • determine experimentally the characteristics of an enzyme, using safe

practices (4.3).

Separation of proteins1.3

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1.3: Separation of proteins

1 Separation techniquesBefore you startIf you find some parts of this unit challenging, remember you are working at a higher level than you may be used to. In this unit it is important that you fully understand the following themes and topics before you begin:

• structure and function of biological molecules • enzyme structure and function • aerobic respiration.

If you need to check your understanding of proteins, carbohydrates, lipids and nucleic acids, Unit 2 Module 1 of OCR AS Biology (P. Kennedy and F. Sochacki, 2008), offers a good introduction to the topic.

If you need to check your understanding of aerobic respiration and the stages of glycolysis, link reaction, the Krebs cycle and the electron transport chain, you may find Unit 1 Module 4 of OCR A2 Biology (S. Hocking, 2008) useful.

Gel filtration chromatographyGel filtration can be used to separate proteins based on their shape and size. It consists of a stationary phase, which is a cross-linked polysaccharide that forms porous beads, and a mobile phase that consists of a buffer. Smaller protein molecules can diffuse easily into the beads and move slowly down the column. Molecules are therefore separated in order of decreasing molecular size (see Figure 1.3.1).

Large particles cannot enter gel and are excluded. They have less volume to traverse and elute sooner.

Small particles can enter gel and havemore volume to traverse. They elutelater.

Chromatogram

Flow

Time

Affinity chromatographyThe mechanism for affinity chromatography is based on adsorption chromatography. Substances can be purified by being attached to a ligand immobilised on a matrix. Protein structure is important in affinity chromatography because ligands used during this process are specific to a protein’s structure. Both the protein and the ligand are covalently bonded to a matrix. Once the process has commenced the target protein becomes trapped on a solid or stationary phase while the other molecules do not become trapped because they do not have the affinity for the ligand (see Figure 1.3.2). The solid medium can finally be removed to release the target protein in a process known as elution.

Figure 1.3.1: Gel filtration chromatography.

Key termsLigand: A molecule that binds to another, larger molecule. In affinity chromatography it is a molecule that binds to a central metal atom to form a coordination complex.

Elution: The process of extracting one material from another.

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The enzyme binds to the immobilisedsubstrate; all other proteins that donot bind to the substrate are eluted inthe void volume of the column

Polyacrylamide gel electrophoresis (PAGE)SDS-PAGE is a method for separating proteins by electrophoresis using a polyacrylamide gel and sodium dodecyl sulfate (SDS) which denatures the proteins. When proteins are mixed with SDS (an anionic detergent) they have a net negative charge. SDS binds to the polypeptide chains in relation to their relative molecular mass. In doing so, it disrupts the structure of the protein and the protein-SDS complexes are strongly attracted towards an anode. The polyacrylamide gel’s structure allows smaller molecules to move further quicker while larger molecules do not move as far (see Figure 1.3.3). By running proteins of known mass at the same time, the relationship between the Rf value (a measurement of how far the compound has moved along the solid support) and mass can be plotted and used to estimate the mass of the unknown samples.

Large,high chargeLarge,low chargeSmall,high chargeSmall,low charge

Vertical slab gelbetween two glass plates

Polyacrylamidepolymer fibres

Anode Cathode

+ –

Ion exchange chromatographyAnother method for the purification of proteins is ion exchange chromatography. During cation exchange chromatography the positively-charged molecules are attracted to a negatively-charged solid support. During anion exchange chromatography, however, negatively-charged molecules are attracted to a positively-charged solid support.

Figure 1.3.2: Affinity chromatography.

Key termRf: The distance moved by the solute divided by the distance moved by the solvent.

Figure 1.3.3: SDS-PAGE electrophoresis.

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The mobile phase used in ion exchange chromatography is a low to medium conductivity solution so there will be a low to medium salt concentration. It is the ionic interactions between the oppositely-charged ionic groups in the sample and in the solid support phase which cause the adsorption of the molecules to the solid support. The charges present in the functional groups of the protein will determine the interaction of the molecule with the support phase.

Take it furtherThere are several suitable books or websites that explain in more detail different separation and purification techniques used in industry. Try Isolation and Purification of Proteins (R. Hatti-Kaul, B. Mattiasson, 2003), Volume 27, Biotechnology and Bioprocessing series, CRC Press.

ChecklistIn this topic you should now be familiar with the following ideas about separation and purification of proteins:

proteins can be separated according to shape and size, relative molecular mass and affinity

gel filtration separates proteins through polysaccharide porous beads and it is based on size

affinity chromatography separates proteins based on their affinity to specific ligands

polyacrylamide gel electrophoresis (PAGE) separates proteins based on relative molecular mass.

Further readingBoyle, M. & Senior, K. (2008) Biology, 3rd Edition, HarperCollins

Campbell, M.K. & Farrell, S.O. (2011) Biochemistry, Cengage Learning

Kennedy, P., Sochacki, F. & Hocking, S. (2008) OCR Biology AS, Heinemann (Pearson Education Limited)

Kennedy, P., Sochacki, F., Winterbottom, M. & Hocking, S. (2008) OCR Biology A2, Heinemann (Pearson Education Limited)

Loomis, H.F. (2005) Enzymes: The Key to Health, 21st Century Nutrition Publishing

Moran, L., Horton, R., Scrimgeour, G., Perry, M. & Rawn, D. (2011) Principles of Biochemistry (International Edition), 5th Edition, Pearson

AcknowledgementsThe publisher would like to thank the following for their kind permission to reproduce their photographs:

Getty Images: Martin McCarthy / E+

All other images © Pearson Education

We are grateful to the following for permission to reproduce copyright material:

Figure 1.3.1: Gel filtration chromatography made by Isaac Yonemoto using Inkscape. Used under the Creative Commons Attribution 2.5 Generic licence; Figure 1.3.2: The Affinity Chromatography Diagram © copyright David A. Bender, http://www.david-bender.co.uk. Used by permission of David A. Bender.

In some instances we have been unable to trace the owners of copyright material, and we would appreciate any information that would enable us to do so.

LinkDNA and mRNA isolation are also discussed in Unit 7: Molecular biology and genetics.

topic guide 1.3: separation of proteins

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