Chapter 3- Exploring proteins Flashcards
Proteome
Derived from proteins expressed by the genome. It encompasses the types, functions, and interactions of proteins within its biological environment. Only a subset of the proteins encoded by genes will actually be present in a given biological context
Is a proteome a fixed characteristic of the cell?
No, it represents the functional expression of information, and it varies with cell type, developmental stage, and environmental conditions. It is highly dynamic
Why is the proteome larger than the genome?
Almost all gene products are proteins that can be chemically modified in a variety of ways
Purification
Should yield a sample containing only one type of molecule
Assay
A test. Biochemists use assays for some unique identifying property of the protein to monitor the success of purification. A positive result on the assay indicates that the protein is present. The more specific the assay, the more effective the purification
How are enzymes detected in a sample?
The assay usually measures enzyme activity, or the ability of the enzyme to promote a specific chemical reaction. This is done indirectly. Lactate dehydrogenase activity is measured by examining how much light absorbing ability is developed by the sample in a given period of time. Molecules produced by the reaction have differing abilities to absorb light
Homogenate
A mixture of all of the components of the cell, after the cell has been disrupted in a homogenizer
What has to happen to the cell before the protein can be purified?
The protein has to be released from the cell. A homogenate is formed by disrupting the cell membrane, and the mixture is fractionated by centrifugation and different proteins can be separated based on density
Differential centrifugation
The mixture is fractionated by a centrifuge, and creates a dense pellet of heavy material at the bottom of the tube with a lighter supernatant above. The supernatant can then be centrifuged at a greater force to create another pellet and supernatant. It yields multiple fractions of decreasing density, with each one containing hundreds of proteins. The fractions are each separately assayed for the desired activity
Salting out
Most proteins are less soluble at high salt concentrations. The salt concentration at which a protein precipitates differs from one protein to another. This means that salting out can be used to fractionate proteins and to concentrate dilute solutions of proteins
Dialysis
Uses a semipermeable membrane (like cellulose with pores) to separate proteins from small molecules like salt. The protein mixture is placed inside the dialysis bag, which is then submerged in a buffer solution that lacks the small molecules to be separated away. Molecules with dimensions greater than that of the pore diameter are left inside the bag, and smaller molecules and ions can pass through the pores and leave the bag, moving down their concentration gradient. It separates small molecules from a cell fractionate, but does not distinguish between proteins
Gel-filtration (molecular exclusion) chromatography
Separates proteins on the basis of size. The sample is added to the top of a column consisting of porous beads made of an insoluble polymer. Small molecules can enter the beads, but large ones can’t. The small molecules are distributed inside the beads and between them, but large molecules flow more rapidly through the column and emerge first. Molecules of medium size will occasionally enter the beads and will leave at an intermediate position, while small molecules take a longer path and exit last
Ion-exchange chromatography
Separates proteins on the basis of their net charge. A protein with a net positive charge will usually bind to a column of beads containing carboxylate groups. A negatively charged protein will not. The bounds protein can then be eluted (released) by increasing the concentration of sodium chloride in the eluting buffer. Sodium ions compete with positively charged groups on the protein for binding to the column. Proteins that have a low density of net positive charge will tend to emerge first (those with the same charge as the column), followed by those with a higher charge density
Cation exchange
Another name for ion exchange chromatography. It indicates that positively charged groups will bind to the anionic beads. Positively charged proteins are considered cationic and are separated by chromatography on negatively charged CM-cellulose columns.
Anion exchange
Negatively charged (anionic) proteins are separated on positively charged DEAE-cellulose columns.
Affinity chromatography
Takes advantage of the high affinity of proteins for specific chemical groups. The protein will pass through a column of beads containing residues of the compound with the affinity for that protein. This is a technique that can be used to isolate transcription factors. The protein mixture is passed through a column that contains specific DNA sequences attached to a matrix. Proteins with a high affinity for the sequence will bind and be retained. The transcription factor is released by washing with a solution that has a high concentration of salt, or with a solution that has a high concentration of the compound to which the protein is bound
Transcription factors
Proteins that regulate gene expression by binding to specific DNA sequences
High performance liquid chromatography
In column chromatography, a solvent drips through a column filled with an adsorbent under gravity. HPLC is a highly improved form of column chromatography. A pump forces a solvent through a column under high pressures. The column is made of finer materials, so they have more interaction sites and more resolving power. Pressure, using high pressure pumps, has to be applied to the column to obtain adequate flow rates. This causes high resolution and rapid separation. The components of a mixture are separated from each other due to their different degrees of interaction with the absorbent particles. This causes different elution rates for the different components and leads to the separation of the components as they flow out the column.
Gel electrophoresis
Uses an electric field to separate molecules with net charges, like proteins, DNA, and RNA. It is performed in a thin, vertical slab of polyacrylamide gel. All molecules, regardless of size, are forced to move through the gel due to the electric field. The proteins are prepared using SDS. Proteins in the sample are separated on the basis of mass. Small proteins move through the gel rapidly, but large proteins stay near the top of the gel.
Why is polyacrylamide gel used in gel electrophoresis?
The gels are chemically inert and readily formed by the polymerization of acrylamide with a small amount of the cross linking agent methylenebisacrylamide to make a 3D mesh.
SDS
Before electrophoresis, the proteins are dissolved in a solution of SDS, which is an anionic detergent that disrupts almost all noncovalent interactions in the proteins. Beta-mercaptoethanol is also used to reduce disulfide bonds. Anions of SDS bind to the main protein chains and give the protein a negative charge that is much greater than the charge of the native protein, but the charge is proportional to the mass of the denatured protein
How are proteins visualized in gel electrophoresis?
They are visualized by staining with silver nitrate or Coomassie blue dye, which creates a series of bands
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Rapid, sensitive, and capable of a high degree of resolution. Even a very small amount of a protein can be stained and create a band. Proteins that differ in mass by about 2% can usually be distinguished with SDS-PAGE. SDS denatures proteins, and 1 molecule of SDS binds for every 2 amino acids, giving every protein the same charge-mass ratio so all proteins will move on the basis of mass only
Velocity of proteins migrating in an electric field
v= Ez/f
E is electric field strength, z is the net charge on the protein, and f is the frictional coefficient. V is inversely proportional to f
Frictional coefficient of proteins migrating in an electric field
f= 6(pi)(eta-n)(r)
Eta (n) is the viscosity of the medium
r is the radius of a sphere
Electrophoretic mobility
Increases for more highly charged solutes and for solutes of smaller size. The electrophoretic mobility of many proteins in SDS–polyacrylamide gels is inversely proportional to the logarithm of their mass. The graph has a negative slope and is linear
Isoelectric point
The pH at which the protein’s net charge is zero. At this pH, the electrophoretic mobility is zero
Isoelectric focusing
Separating proteins on the basis of their isoelectric point. Each protein will move until it reaches a position in the gel at which the pH is equal to the pI of the protein, or the point at which their charge is zero. In electrophoresis, a molecule will always stop moving once it has no charge. This method can resolve proteins that differ in pI by as little as .01, so proteins differing by one net charge can be separated.
How is the pH gradient formed in the isoelectric focusing gel?
By subjecting a mixture of polyampholytes (small multicharged polymers) having many different pI values to electrophoresis
Amino acids containing carboxyl groups are uncharged when
Protonated- Asp and Glu are examples
Amino acids containing nitrogen groups are uncharged when
Deprotonated- Lys, His, and Arg are examples
Two dimensional electrophoresis
Isoelectric focusing combined with SDS-PAGE. A sample is first subjected to isoelectric focusing. The single lane gel is placed horizontally on top of an SDS-polyacrylamide slab. The proteins are therefore spread across the top of the polyacrylamide gel according to how far they migrated during isoelectric focusing (on the basis of their isoelectric point). Then, they undergo electrophoresis again in a vertical direction to yield a 2D pattern of spots. The proteins spread in the vertical direction on the basis of mass
2D gel electrophoresis is typically used for
Proteins isolated from cells under different physiological conditions. The intensities of the individual spots on the gels can be compared, indicating the concentrations of specific proteins have changes in response to the physiological state
How is a protein purification scheme quantitatively evaluated? (5)
Measured by calculating the specific activity after each protein purification technique. The sample then undergoes SDS-PAGE. The techniques analyzed includes homogenization, salt fractionation, ion exchange chromatography, gel filtration chromatography, and affinity chromatography. Specific activity should increase with each step of the purification procedure since total protein is being removed while desired enzyme is being retained. SDS-PAGE allows a visual evaluation of the purification scheme
Specific activity
The ratio of enzyme activity to total protein concentration. Specific activity should increase with each step of the purification procedure since total protein is being removed while desired enzyme is being retained
Zonal (gradient) centrifugation
Used to separate proteins with different sedimentation coefficients. A density gradient is formed in a centrifuge tube, using differing proportions of a low density solution and a high density solution, like 5% and 20% sucrose. The solutions are mixed and create a linear gradient of sucrose concentration, which is highest at the bottom of the tube and lowest at the top. It stabilizes the sedimentation of the molecules. On centrifugation, particles move away from the starting zone with velocities determined both by their size and shape and by the centrifugal force to which they are subjected. After centrifugation for a certain time, particles will be found in a series of zones spaced according to the relative velocities of the particles. In this way particles differing in sedimentation rate by 20% or less can be separated without undue difficulty. Rate-zonal centrifugation thus complements differential centrifugation. The separated bands (zones) of proteins can be harvested by making a hole in the bottom of the tube and collecting drops
Sedimentation coefficients
Usually expressed in Svedberg units (S). The smaller the S value, the more slowly a molecule moves in a centrifugal field
What are the protein drops collected during zonal centrifugation analyzed for?
Protein content, catalytic activity, other biochemical characteristics or functional properties
Advantages of recombinant technology (3)
- Proteins can be expressed in large quantities
- Affinity tags can be fused to proteins that allow
purification of the protein or visualization of the protein in the cell - Proteins with modified primary structures can be readily generated
Antibody
Also called an immunoglobulin. A protein synthesized by vertebrates in response to the presence of an antigen. Antibodies are specific and have a high affinity for the antigens that elicited their synthesis. The binding of an antibody to an antigen is a step in the immune response that protects the animal from infection
Antigen
A foreign substance, antibodies are synthesized in response to an antigen. Foreign proteins, polysaccharides, and nucleic acids can be antigens. Small foreign molecules like synthetic peptides can also act as antigens if the small molecule is attached to a macromolecular carrier
Antigenic determinant (epitope)
A specific group or cluster of amino acids on the target molecule that can be recognized by an antibody
Antibody structure
Antibodies can contain multiple chains, linked by disulfide bonds. One antibody can have multiple domains and multiple antigen binding sites. For example, IgG has 3 domains (2 Fab domains and 1 Fc domain). There is an antigen binding site on each Fab domain
Antibody-antigen interactions IgG
A protein antigen binds to the end of an Fab domain of an antibody. The end of the antibody and the antigen have complementary shapes, which allows a large amount of surface to buried when the two proteins bind
Polyclonal antibodies
Antibodies derived from multiple antibody-producing cell populations. Each recognizes a different surface feature of the same antigen. They are advantageous for the detection of a protein of low abundance, because each protein molecule can be bound by more than one antibody at multiple distinct antigenic sites
