Bio-479 Chapter 5

  1. In protein sequencing, the protein is typically degraded into smaller peptides which are sequenced and the full-length protein sequence is then reconstructed by assembling the overlapping fragments. 

    True or False ?
    True
  2. The amino acid sequence of a polypeptide can be determined through repetitive cycles of the _____
    Edman Degradation
  3. Which technique does NOT separate proteins on the basis of size?

    Ultracentrifugation.
    Ion-exchange chromatography.
    Gel filtration chromatography.
    SDS-Polyacrylamide gel electrophoresis.
    Ion-exchange chromotography
  4. In two-dimensional (2D) gel electrophoresis, a sample of proteins is first subjected to isoelectric focusing, and then to SDS-PAGE in a perpendicular direction.

    True or False ?

    True
  5. A protein is most soluble at its isoelectric point. ?

    True or False
    False
  6. The aromatic residues phenylalanine, tyrosine and tryptophan are the three amino acids responsible for proteins high absorption at the 280 nm wavelength, therefore measuring a sample's UV absorbance at 280 nm gives an accurate measurement of its protein content.

    True or False ?
    False -Correct. Since many substances that are likely to be present during a protein purification procedure (e.g., nucleic acids) also absorb UV light, and since proteins vary in their proportion of light-absorbing aromatic residues, spectroscopic measurements in the UV region can only provideestimates of the amount of protein present (although they can accurately determine the amount of a pure protein of known molar absorptivity.
  7. Temperature and pH are important factors to consider during the purification of proteins. 

    True or False ?
    True
  8. ________ uses a stable pH gradient to separate proteins.
    Isoelectric focusing
  9. Affinity chromatography -

    Separation on the basis of size and shape.

    Separation on the basis of polarity.

    Separation on the basis of charge.

    Separation on the basis of binding specificity.
     
    Separation on the basis of binding specificity.
  10. Hydrophobic interaction chromatography

    Separation on the basis of binding specificity.
    Separation on the basis of size and shape.
    Separation on the basis of polarity.
    Separation on the basis of charge.
    Separation on the basis of polarity.
  11. Ion-exchange chromatography

    Separation on the basis of size and shape.

    Separation on the basis of charge.

    Separation on the basis of binding specificity.

    Separation on the basis of polarity.
    Separation on the basis of charge.
  12. Only a tiny fraction of all possible proteins exist (i.e. of all combinations of protein sequence possible, evolution has produced only a tiny fraction of the theoretical possibilities).

    True or False ?
    True
  13. Short polypeptides (<25 residues) can be directly sequenced though the use of a tandem mass spectrometer. The first mass spectrometer functions to select and separate the peptide ion of interest from peptide ions of different masses as well as any contaminants that may be present. The selected peptide ion is then fragmented predominantly at only one of its several peptide bonds, thereby yielding one or two charged fragments per original ion. The molecular masses of the numerous charged fragments so produced are then determined by the second mass spectrometer. By comparing the molecular masses of successively larger members of a family of fragments, the molecular masses and therefore the identities of the corresponding amino acid residues can be determined.

    True or False ?
     
    True
  14. Which of the structures shown below is 1-dimethylaminonaphthalene-5-sulfonyl chloride (also called dansyl chloride) a reagent which reacts with primary amines to yield a fluorescent compound.

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  15. In gel filtration chromatography, molecules are separated according to their size and shape where larger molecules elute first and smaller molecules elute last.

    True of False ?
    • True -
    • Correct. In gel filtration chromatography, molecules are separated according to their size and shape. The stationary phase consists of gel beads containing pores where the pores function as “molecular sieves" with the molecules that are too large to pass through the pores being excluded from the solvent volume inside the gel beads. These large molecules therefore are eluted first and smaller molecules that do pass through the pores are eluted last.
  16. Which chemical reagent promotes peptide bond cleavage on the C-side (carboxyl side) of lysine and arginine residues if the next residue is not proline?

    cyanogen bromide
    trypsin
    performicacid
    chymotrypsin
    phenylisothiocyanate
    elastase
    2-mercaptoethanol
    Trypsin promotes the cleavage of peptide bond cleavage on the C-side of Lys and Arg residues if the next residue is not proline.
  17. In normal electrophoresis the molecular separations are based on gel filtration (their size and shape) as well as electrophoretic mobility (their electric charge). In SDS-PAGE however, SDS-treated proteins have similar shapes and charge-to-mass ratios. SDS-PAGE therefore separates proteins purely by gel filtration effects, that is, according to molecular mass.  

    True of False ?
    True
  18. Which chemical reagent promotes peptide bond cleavage on the C-side (carboxyl side) of Met residues?

    chymotrypsin
    2-mercaptoethanol
    elastase
    cyanogen bromide
    performic acid
    trypsin
    phenylisothiocyanate
    cyanogen bromide
  19. Which reagent reduces disulfide bonds to sulfhydryl groups?

    iodoacetate
    performic acidcyanogen bromide (CNBr)
    dansyl chloride
    2-mercaptoethanol
    2-mercaptoethanol
  20. Which reagent cleaves disulfide bonds oxidatively?

    2-mercaptoethanol
    iodoacetatecyanogen bromide (CNBr)
    dansyl chloride
    performic acid
     
    performic acid
  21. The first protein sequenced, reported by Frederick Sanger, was bovine ________ 
     
    Insulin
  22. he addition of ions weakens ionic interactions between proteins, leading to greater solubility, but too many ions can deprive the protein of solvent leading to protein precipitation is ____
    Salting out
  23. The phenomenon in which protein solubility is increased as salt is added is called salting out 

    True or False ?
    False

    Salting out is the phenomenon in which protein solubility is decreased as salt is added.
  24. One of the keys to deciphering the function of a given protein is to understand its structure.

    True or False?

     
    False ,

    Protein function is related to its structure because a protein needs to assume a particular structure for it to function correctly.
  25. Which statement(s) is/are TRUE about the following peptide? Ala-Cys-Gly-Met-Lys

    It has two positive charges
    It has four peptide bonds
    It has a disulphide bridge
    • It has two positive charges
    • It has four peptide bonds
  26. You are trying to purify a protein that is soluble in a solution of 2 M ammonium sulfate. After centrifugation to remove other proteins that have precipitated at this high salt concentration, you recover the supernatant to assay the target protein's activity in a cell culture system. The cells die when incubated with the supernatant because they  lose water to the high salt solution.gain__________. What procedure might you perform to correct the problem______
    lose water to high salt solution

    fractionation
  27. Insulin (Fig. 5-1) is treated with mercaptoethanol to break its disulfide bonds. The mercaptoethanol is removed so that disulfide bonds can re-form. Since there are (type the number)___ Cys residues on the A chain and ____ on the B chain, there are a total of____ possible ways to form a single Cys-Cys linkage between the two chains. For two disulfide bonds, there are _____ ways of forming the first bond and then____ ways of forming the second bond for a total of ____  possible ways to form two disulfide bonds.There are therefore ___ different ways to linking the A and B chains via disulfide bonds..
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Bio-479 Chapter 5
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