Ch 3 Text 2

  1. ·         Proteins can be separated by __and displayed.
    o   How can we tell that a purification scheme is effective? One way is to ascertain that the __ __ with each purification step. Another is to determine that the number of different proteins in each sample __at each step, which can be done in gel electrophoresis
    gel electrophoresis 

    specific activity rises

  2. o   Gel electrophoresis: a molecule with a __ will move in an __. The __of a protein in an electric field depends on the __, __, and __.
    • net charge
    • electric field
    • velocity of migration (v) 
    • electric field strength (E), the net charge on the protein (z), and the frictional coefficient (f).
  3. §  V=Ez/f: The electric force Ez driving the __toward the __ is opposed by the __arising from friction between the moving molecule and the medium. The frictional coefficient f depends on both the __ and _ of the __ and the __ of the __. For a sphere of radius r, f=6Πη
    • charged molecule 
    • oppositely charge electrode
    • viscous drag fv 
    • mass and shape of the migrating molecule and the viscosity (η) of the medium
  4. §  These separations are nearly always carried out in __because the gel serves as a molecule sieve that does what?
    · Molecules that are small compared with the pores in the gel do what, whereas molecules much larger than the pores are __
    §  The electric field causes proteins to do what?
    • porous gel 
    • enhances separation
    • readily move through the gel
    • almost immobile
    • migrate from the negative to the positive
  5. §  This is performed in __ because of its __and __ by the __ of __ with a small amount of the cross-linking agent __to make a __
    §  Electrophoresis is different from gel filtration in that, __
    • polyacrylamide gel
    • inertness 
    • ready formation
    • polymerization of acrylamide
    • methylenebisacrylamide 
    • 3D mesh
    • because of the electric field, all of the molecules are forced to move through the same matrix
  6. §  Proteins can be separated largely on the basis of mass by __under denaturing conditions
    · Protein mixture first dissolved in a solution of __, an anionic detergent that disrupts nearly all noncovalent interactions in native proteins 
    electrophoresis in a polyacrylamide gel 

    dodecyl sulfate (SDS)
  7. · __ or __ is added to reduce __; and, anions of SDS bind to __ at a ratio of about __. The negative charge acquired on binding SDS is usually much greater than the __; the contribution of the protein to the total charge of the SDS- protein complex is thus rendered insignificant
    • Beta-mercaptoethanol or dithiothreitol
    • disulfide bonds
    • main chains
    • one SDS anion for every two amino acid residues
    • charge on the native protein
  8. o   As a result, this complex of SDS with a denatured protein has a large __that is roughly proportional to the mass of the proteins.
    o   The SDS-protein complexes are then subjected to __
    • net negative charge 
    • electrophoresis
  9. · Completion of electrophoresis--> __--> bands
    · Radioactive labels are also used in a process called __
    §  Small proteins move rapidly through the gel, whereas large proteins stay at the top
    proteins visualized by staining them with silver or a dye like Coomassie blue

  10. · The mobility is linearly proportional to the __. Some carb-rich proteins and membrane proteins do not obey this
    o   __ is rapid, sensitive, and capable of a high degree of resolution
    §  Small portions can still be detected
    • logarithm of their mass
    • SDS-polyacrylamide gel electrophoresis
  11. §  The initial fractions will display dozens to hundreds of proteins. As the purification progresses, what happens?
    o   Isoelectric focusing: Electrophoresis also separates on the basis of __ and __. The isoelectric point of a protein is the __
    §  At this pH, its electrophoretic mobility is __because z in equation 1 is equal to zero
    the number of bands will diminish and the prominence of one of the bands should increase

    acidic and basic residues

    pH at which its net charge is zero

  12. · This method of separating proteins according to their isoelectric point is called __. The pH gradient in the gel is formed first by subjecting a mixture of __(small multicharged polymers) having many different pI values to electrophoresis
    • isoelectric focusing
    • polyampholytes
  13. o   Two-dimensional electrophoresis
    §  A single sample is first subjected to __. This single-lane gel is then placed __on top of an __. The proteins are thus spread across the top of the polyacrylamide gel according to __. Then they undergo __again in a __to yield a 2D pattern of spots. In such a gel, proteins have been separated in the horizontal direction on the basis of __ and in the vertical direction on the basis of __.
    • isoelectric focusing
    • horizontally 
    • SDS-polyacrylmide slab
    • how far they migrated during isoelectric focusing
    • electrophoresis 
    • perpendicular direction (vertically) 
    • isoelectric point
    • mass
  14. o   A protein purification scheme can be quantitatively evaluated
    §  To determine the success of a protein purification scheme, we monitor each step of the procedure by determining the __ and subjecting it to __
    specific activity of the protein mixture

    SDS-PAGE analysis
  15. Total Protein
    the quantity of protein present in a fraction is obtained by determining the protein concentration of a part of each fraction and multiplying by the fraction’s total volume
  16. Total Activity
    The enzyme activity for the fraction is obtained by measuring the enzyme activity in the volume of fraction used in the assay and multiplying by the fraction’s total volume
  17. Specific Activity
    · This parameter is obtained by dividing total activity by total protein
  18. Yield
    · This parameter is a measure of the activity retained after each purification step as a percentage of the activity in the crude extract. The amount of activity in the initial extract is taken to be 100%
  19. Purification Level
    · Purification Level: this is a measure of the increase in purity and is obtained by dividing the specific activity, calculated after each purification step, by the specific activity of the initial extract
  20. · After __to lower the high concentration of salt remaining from the __, the fraction is passed through an __
    • salt fractionation
    • dialysis 
    • salt fractionation
    • ion-exchange column
  21. · The purification now increases to__ compared with the original extract, whereas the yield falls to 77%
    · __ brings the level of purification to __, but the yield is now at 50%.
    · The final step is __with the use of a ligand specific for the target enzyme
    o   This is the most powerful step and results in a purification level of __ but lowers the yield to 35%
    • 9-fold
    • Gel filtration chromatography 
    • 110-fold
    • affinity chromatography 
    • 300-fold
  22. §  If we load a constant amount of protein onto each lane after each step, the number of bands __in proportion to the __, and the amount of protein of interest __as a __
    §  A good purification scheme takes into account both __ and __. How so?
    • decreases 
    • level of purification
    • increases 
    • proportion of the total protein present
    • purification levels and yield
    • A high degree of purification and a poor yield leave little protein. A high yield with low purification leaves many contaminants in the fraction and complicates things
  23. o   __is valuable for separating biomolecules and determining their masses
    §  Centrifugingà mass and density, shape, interactions can be determined but with the use of a __; this force enables particles to __. To quantify the rate of movement,__, of a particle can be determined with s= m(1-vp)/f, where m is the mass, v is the partial specific volume, p is the density of the medium, and f is the frictional coefficient (a measure of the shape of the particle). The (1-vp) term is the __ exerted by __
    • Ultracentrifugation 
    • mathematical description of how a particle behaves when a centrifugal force is applied
    • move through liquid media
    •  s, the sedimentation coefficient
    • buoyant force
    • liquid medium
  24. o   Sedimentation coefficients are usually expressed in __units, equal to 10-13 s. The smaller the S value, the __.
    o   Several important conclusions can be drawn from the preceding equation:
    §  The sedimentation velocity of a particle depends in part on its __. Explain.
    §  __affects the __. The frictional coefficient f of a compact particle is __than that of an extended particle of the same mass. __ particles sediment more slowly
    • Svedberg 
    • more slowly a molecule moves in a centrifugal field
    • mass. A more massive particle sediments faster than a lesser one
    • Shape 
    • viscous drag
    • smaller 
    • Elongated
  25. §  A dense particle moves more rapidly than does a less dense one because what?
    §  The sedimentation velocity also depends on the __of the solution. Particles sink when __, float when__, and do not move when it equals one.

    • the opposing buoyant force is smaller for the denser particle
    • density 
    • vp < 1
    •  vp > 1
  26. o   A technique called __, __, or __ can be used to separate proteins with __.
    §  Step One: __
    · Different amounts of low-density and high-density solutions are mixed to create a __ of __ ranging from 20% at the bottom of the tube to 5% at the top
    • zonal, band, or gradient centrifugation
    • different sedimentation coefficients
    • form density gradient in tube
    • linear gradient of sucrose concentration
  27. o   The role of the gradient is to prevent __
    o   A small volume of a solution containing the mixture of proteins to be separated is placed wheree?

     When the rotor is spun, proteins move through the gradient and separate according to their __. The time and speed is determined __. The separated bands, or zones, of protein can be harvested by __, which can then be __. This __ technique readily separates proteins differing in __ by a factor of two or more
    • convective flow
    • on top of the density gradient
    • sedimentation coefficients
    • empirically
    • by making a hole in the bottom of the tube and collecting drops
    • measured for protein content and catalytic activity or another functional property
    • sedimentation-velocity
    • sedimentation coefficient
  28. · The mass of a protein can directly be determined by __, in which a sample is centrifuged at low speed such that a __ of the sample is formed. However, this sedimentation is counterbalanced by the __.. 
    • sedimentation equilibrium
    • concentration gradient
    • diffusion of the sample from regions of high to low concentration
  29. o   When equilibrium is achieved, the shape of the final gradient depends only on the __
    §  The __ for determining mass is very accurate and can be applied without __the protein. Thus the native quaternary structure of multimeric proteins is preserved
    · In contrast, __provides an estimate of the mass of dissociated polypeptide chains under __conditions
    • mass of the sample
    • sedimentation-equilibrium technique
    • denaturing 
    • SDS-polyacrylamide gel electrophoresis 
    • denaturing
  30. • If we know the mass of the dissociated components of a __ as determined by __ and the __ of the __ as determined by __, we can determine the number of copies of each polypeptide chain present in the protein complex
    • multimeric protein
    • SDS-polyacrylamide analysis
    • mass of the intact multimer
    • sedimentation-equilibrium analysis
  31. o   Protein purification can be made easier with the use of __
    §  Before these techniques, proteins were isolated solely from their __, often requiring a large amount of tissue to obtain a sufficient amount of protein for study
    • recombinant DNA technology
    • native sources
  32. §  Now, they are advantaged in a number of ways:
    What are the three
    Proteins can be expressed in large quantities

    affinity tags can be fused to proteins

    proteins with modified primary strutures can  be readily generated
  33. Proteins can be expressed in large quantitieshomogenate
    ·  The __serves as the starting point in a protein purification scheme. For recombinant systems, a host organisms that is amenable to __ is utilized to express the protein of interest. Purification can begin with a __that is often __. A protein can also easily be obtained regardless of its natural abundance or species of origin
    • homogenate
    • genetic manipulation
    • homogenate 
    • highly enriched with the desired molecule
  34. · Affinity tags can be fused to proteins: __ enables the attachment of any one of a number of possible affinity tags to a protein. Hence, the benefits of __ can be realized even for those proteins for which a binding partner is unknown or not easily determined
    • Recombinant DNA
    • affinity chromatography
  35. · Proteins with modified primary structures can be readily generated: __ can manipulate genes to do what? With the use of genetic-manipulation strategies, __ can be generated. Additionally, __ can be introduced into the active site of an enzyme to precisely __the roles of the specific residues within its __
    • recombinant DNA technology
    • generate variants of a native protein sequence
    • fragments of a protein that encompass single domains 
    • amino acid substitutions
    • probe 
    • catalytic cycle
Card Set
Ch 3 Text 2
Test One