Chapter 5 Techniques In Protein Biochemistry

Front 1

refers to the proteins expressed by the genome

Back 1

proteome

Front 2

encompasses the types, functions, and interactions of proteins that yield a functional unit

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proteome

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varies with cell type, developmental state, and environmental conditions

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proteome

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any procedure to measure the activity of a biomolecule, such as an enzyme

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assay

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mixture formed when a tissue is treated in such a way that the cell membranes are disrupted

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homogenate

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What is salting out?

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protein precipitation caused by an increase in the salt concentration

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process of removing small molecules via diffusion from a mixture of solutes, which is placed in a semipermeable bag placed in a large volume of solvent

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dialysis

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How does gel-filtration chromatography work?

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A sample is applied to the top of a column consisting of porous beads made of an insoluble polymer. Small molecules can enter these beads, but large ones cannot, and so the larger molecules follow a shorter path to the bottom of the column and emerge first. Molecules that are of a size to occasionally enter a bead will flow from the column at an intermediate position, and small molecules, which take a longer, more-circuitous path, will exit last.

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What is another name for gel-filtration chromatography?

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molecular exclusion chromatography

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How does ion-exchange chromatography work?

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A mixture of proteins is percolated through a column of beads that contain a charged group, such as a carboxylate group. Proteins bearing charges opposite those on the bead will bind. The bound proteins can be eluted (released) by washing with a buffer containing salt.

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biochemical purification technique based on a specific interaction between a protein and a ligand

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affinity chromatography

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How does affinity chromatography work?

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A ligand is immobilized on inert material and a protein mixture is passed over the ligand. Only the protein with the specific interaction will bind.

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What does HPLC stand for?

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high-pressure liquid chromatography

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How does high-pressure liquid chromatography work?

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The separation materials are more finely divided and thus possess more interaction sites, resulting in greater resolution.

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Why must pressure be applied in HPLC?

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Because the column material is so fine, it is necessary to obtain adequate flow rates.

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technique for separating biomolecules, such as nucleic acids and proteins, on the basis of charge and size

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gel electrophoresis

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How does gel electrophoresis work?

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When placed under an electric field, biomolecules move through the gel, with larger molecules moving more slowly than smaller molecules.

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What gel is used for electrophoresis?

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a cross-linked polymer such as polyacrylamide

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means of separating proteins electrophoretically based on the relative content of acidic and basic residues of the proteins

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isoelectric focusing

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How does isoelectric focusing work?

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A mixture of proteins undergoes electrophoresis in a pH gradient. Each protein will migrate in the gel until it reaches the pH equal to its pI, the pH at which the protein has no net charge.

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How does two-dimensional electrophoresis work?

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The protein mixture is first separated in one dimension by isoelectric focusing in a single-lane gel. The gel is then placed horizontally on top of an SDS-acrylamide gel and electrophoresis is performed in the second dimension, perpendicular to the first direction.

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How does gradient centrifugation work?

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A linear gradient of viscous solution is created in a centrifuge tube—for instance, a gradient of 5% to 20% sucrose. The sample is layered on the top of the gradient, and the tube is centrifuged at high speeds. Large, dense molecules or complexes move through the gradient faster than smaller complexes. The separated complexes are harvested by making a hole in the bottom of the tube and collecting drops.

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What is another name for gradient centrifugation?

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zonal centrifugation

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rate at which a particle will move in a liquid medium when subjected to centrifugal force

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sedimentation coefficient (s)

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What is the sedimentation coefficient (s) a function of?

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mass, shape, and density of the particle as well as the density of the medium

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What are sedimentation coefficients usually expressed as?

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Svedberg units (S), equal to 10^-13 s

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protein synthesized by an animal in response to a foreign substance

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antibody

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have specific and high affinity for their antigens

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antibodies

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What is a key step in the immune response?

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binding of antibody to antigen

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foreign substance that generates an immune response in an organism

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antigen

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specific group or cluster of amino acids on an antigen that is recognized by an antibody

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antigenic determinant

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What is another name for antigenic determinants?

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epitopes

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produced by a cloned hybridoma cell, binds to only one epitope on a specific antigen

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monoclonal antibody

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similar to affinity chromatography, except here the material bound to the matrix (antibody) has high affinity for the proteins moving over the matrix

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immunoprecipitation

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immunological-based means of detecting and quantifying proteins

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enzyme-linked immunosorbent assay

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What is ELISA?

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enzyme-linked immunosorbent assay

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How does ELISA work?

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An enzyme that reacts with a colorless substrate to produce a colored product is covalently linked to a specific antibody that recognizes a target antigen. If the antigen is present, the antibody-enzyme complex will bind to it; on the addition of the substrate, the enzyme will catalyze the reaction, generating the colored product revealing the presence of the antigen.

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means of detecting small quantities of a protein of interest in a cell or bodily fluid

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western blotting

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How does western blotting work?

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The sample is subject to SDS-gel electrophoresis, and then the proteins in the gel are transferred (blotted) to a polymer sheet pressed against the gel. The blot is then treated with a labeled antibody specific for the protein of interest, allowing for the identification and quantitation of the protein.

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What is another name for western blotting?

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immunoblotting

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protein-sequencing method that sequentially removes one residue at a time from the amino end of a peptide

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Edman degradation

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How does Edman degradation work?

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Phenyl isothiocyanate reacts with the terminal amino group of the peptide, which then cyclizes and breaks off the peptide, yielding an intact peptide shortened by one amino acid. The cyclic compound is a phenylthiohydantoin (PTH) amino acid, which can be identified by chromatographic procedures. The Edman procedure can then be repeated sequentially to yield the amino acid sequence of the peptide.

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chromatographic technique that separates proteins on the basis of size

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gel-filtration

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means of protein purification that takes advantage of the differences in net charge among proteins

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ion-exchange chromatography

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means of obtaining high-resolution separations of proteins in a mixture

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two-dimensional electrophoresis

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enhanced column chromatography technique

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HPLC

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ultracentrifugation technique used to separate large biomolecules or molecular complexes

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gradient centrifugation

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Why is an assay required for protein purification?

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detection of the protein of interest

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What should an assay be based on?

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some unique biochemical property of the protein that is being purified

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What physical differences among proteins allow for their purification?

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Differences in size, solubility, charge, and the specific binding of certain molecules.

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What is the biochemical basis for the power of immunological techniques?

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specificity of the antibody-antigen interaction

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What does the specificity of the antibody-antigen interaction allow for?

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detection of the antigen in the presence of large amounts of extraneous material

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What is amino acid composition?

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amino acids that are present in the protein

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True or false: each protein has a unique amino acid composition.

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false

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the primary structure of a protein

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amino acid sequence

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True or false: each protein has a unique amino acid sequence.

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true

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What is an assay?

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means of identifying a protein based on a unique property of the protein

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What does gel-filtration chromatography do?

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separates proteins on the basis of size differences

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What does ion-filtration chromatography do?

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separates proteins on the basis of net charge

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What is affinity chromatography based on?

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attraction to a specific chemical group or molecule

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What does high-pressure liquid chromatography allow?

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high resolution and rapid separation

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What is isoelectric focusing based on?

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the fact that proteins have a pH at which the net charge is zero

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What is the sedimentation coefficient?

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measure of the rate of movement due to centrifugal force

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What is an epitope?

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specific site recognized by an antibody

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What are monoclonal antibodies produced by?

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hybridoma cells

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What is western blotting?

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an immunoassay technique preceded by gel electrophoresis

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Why do proteins precipitate at high salt concentrations?

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If salt concentration becomes too high, the salt ions interact with the water molecules. Eventually, there are not enough water molecules to interact with the protein, and the protein precipitates.

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Why do some proteins require salt to dissolve?

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If there is a lack of salt in a protein solution, the proteins may interact with one another—the positive charges on one protein with the negative charges on another or several others. Such an aggregate becomes to large to be solubilized by water alone. If salt is added, it neutralizes the charges on the proteins, preventing protein-protein interactions.

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What types of R groups would compete with salt ions for water of solvation?

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Charged and polar R groups on the surface of an enzyme.

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The octapeptide AVGWRVKS was digested with the enzyme trypsin, which cleaves after arginine and lysine. Would ion-exchange or gel-filtration chromatography be most appropriate for separating the products? Explain.

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Gel filtration because AVGWR, VK, and S differ in size.

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The octapeptide AVGWRVKS was digested with the enzyme chymotrypsin, which cleaves after large aliphatic or aromatic R groups. Would ion-exchange or gel-filtration chromatography be most appropriate for separating the products? Explain.

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Ion-exchange because AVGW is neutral and RVKS has two positive charges.

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What does sodium dodecyl sulfate do?

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denatures proteins

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How does SDS destroy protein structure?

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The long hydrophobic tail on the SDS molecule disrupts the hydrophobic interactions in the interior of the protein. The protein unfolds, with the hydrophobic R groups now interacting with SDS rather than with one another.

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In the course of purifying an enzyme, a researcher performs a purification step that results in an increase in the total activity to a value greater than that present in the original crude extract. Explain how the amount of total activity might increase.

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An inhibitor of the enzyme being purified might have been present and subsequently removed by a purification step. This removal would lead to an apparent increase in the total amount of enzyme present.

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How is specific activity calculated?

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total activity/total protein (units/mg)

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How is purification level calculated?

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specific activity/specific activity of crude extract

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How is yield calculated?

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total activity/total activity of crude extract (%)

Front 78

Proteins treated with a sulfhydryl reagent such as beta-mercaptoethanol and dissolved in sodium dodecyl sulfate have the same charge-to-mass ratio. Explain.

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Because one SDS molecule binds to a protein for every two amino acids in the proteins, in principle, all proteins will have the same charge-to-mass-ratio. For instance, a protein consisting of 200 amino acids will bind 100 SDS molecules, whereas a protein consisting of 400 amino acids will bind 200 SDS molecules. The average mass of an amino acid is 110, and there is one negative charge per SDS molecule. Thus, the charge to mass ratio of both proteins is the same—0.0045.

Front 79

Under what conditions will proteins treated with a sulfhydryl reagent such as beta-mercaptoethanol and dissolved in sodium dodecyl sulfate not have the same charge-to-mass ratio?

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if the protein contains many charged amino acids

Front 80

What unique property of the estrogen receptor allows for its identification and purification?

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The estrogen receptor has a unique, high affinity for the estrogen estradiol.