Chapter 5

  1. An assay identifies the desired protein. The ability to identify the protein is important in determining if particular purification steps are effective in isolating the protein from the other cellular material.

  2. Complete the interactive matching exercise to see answers.

  3. If the 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.

  4. 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 too large to be solubilized by water alone. If salt is added, it neutralizes the charges on the proteins, preventing protein–protein interactions.

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

    1. Trypsin cleaves after arginine (R) and lysine (K), generating AVGWR, VK, and S. Because they differ in size, these products could be separated by molecular exclusion chromatography.

    2. Chymotrypsin, which cleaves after large aliphatic or aromatic R groups, generates two peptides of equal size (AVGW) and (RVKS). Separation based on size would not be effective. The peptide RVKS has two positive charges (R and K), whereas the other peptide is neutral. Therefore, the two products could be separated by ion-exchange chromatography.

    C5

  7. 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.

  8. 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.

  9. Purification procedure

    Total protein (mg)

    Total activity (units)

    Specific activity (units mg−1)

    Purification level

    Yield (%)

    Crude extract

    20,000

    4,000,000

      200

    1

    100

    (NH4)2SO4 precipitation

      5,000

    3,000,000

      600

    3

      75

    DEAE–cellulose chromatography

      1,500

    1,000,000

      667

      3.3

      25

    Molecular exclusion chromatography

        500

      750,000

    1,500

      7.5

      19

    Affinity chromatography

          45

      675,000

    15,000

    75

      17

    1. 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.

    2. The statement might be incorrect if the protein contains many charged amino acids.

    3. The protein may be modified. For instance, serine, threonine, and tyrosine may have phosphoryl groups attached.

  11. The term is the partial specific volume, the reciprocal of the particle density. Thus, the denser a particle, the smaller . The smaller means that the opposing force is less, so the denser particle moves faster.

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

  13. Polyclonal antibodies are a collection of antibodies that bind to multiple epitopes on an antigen. Monoclonal antibodies constitute a collection of antibodies that bind to a single epitope on an antigen.

  14. If an antibody to a protein of interest exists, the antibody can be attached to an insoluble bead of some sort. A mixture of proteins that includes the protein of interest is mixed with the antibody. Only the protein of interest will bind to the antibody. The mixture is centrifuged, and the supernatant is discarded. The protein of interest is then released from the antibody, often by adding a protein denaturant.

  15. An enzyme-linked immunoabsorbant assay. ELISA is used for quantitating the presence of an antigen by using an enzyme linked to an antibody to the antigen.

  16. Western blotting is an immunological technique used to detect a specific protein in a cell or in a body fluid. A sample is subjected to SDS–polyacrylamide electrophoresis. The resolved proteins are transferred, or blotted, to a polymer sheet, and then an antibody specific for the protein of interest is incubated with the blotted sample. Other, enzyme-linked antibodies can then be used to visualize the desired antigen–antibody complex.

  17. Keep in mind that we are sequencing a large population of identical molecules, not a single molecule, and that the cleavage reaction is not 100% effective. Consequently, after many repetitions (approximately 50), many different peptides are releasing different amino acids at the same time. To illustrate this point, assume that each sequencing step is 98% efficient. The proportion of correct amino acids released after 50 rounds is 0.9850, or 0.4—a hopelessly impure mix.

  18. Many proteins have similar masses but different sequences and different patterns when digested with trypsin. The set of masses of tryptic peptides forms a detailed “fingerprint” of a protein that is very unlikely to appear at random in other proteins regardless of size.

  19. Treatment with urea disrupts noncovalent bonds. Thus, the original 60-kDa protein must be made of two 30-kDa subunits. When these subunits are treated with urea and β-mercaptoethanol, a single 15-kDa species results, suggesting that disulfide bonds link the 30-kDa subunits.

    1. At the pI, the protein has no net charge, so the repulsive forces between protein molecules are minimal. This lack of repulsion allows individual proteins to interact, forming large complexes that cannot be solvated; that is, the complexes precipitate.

    2. Salt binds to the charges on the protein, preventing protein-protein interactions that lead to precipitation. This is the process of salting in (Figure 5.2 and problem 4).

  21. Amino terminal: A

    Trypsin digestion: Cleaves at R. Only two peptides are produced. Therefore, one R must be internal and the other must be the carboxyl-terminal amino acid. Because A is amino terminal, the sequence of one of the peptides is AVR.

    Carboxypeptidase digestion: No digestion confirms that R is the carboxyl-terminal amino acid.

    Chymotrypsin digestion: Cleaves only at Y. Combined with the preceding information, chymotrypsin digestion tells us that the sequences of the two peptides are AVRY and SR.

    Thus, the complete peptide is AVRYSR.

  22. First amino acid: S

    Last amino acid: L

    Cyanogen bromide cleavage: M is 10th position; carboxyl-terminal residues are (2S,L,W).

    Amino-terminal residues: (G,K,S,Y), tryptic peptide, ends in K

    Amino-terminal sequence: SYGK

    Chymotryptic peptide order: (S,Y), (G,K,L), (F,I,S), (M,T), (S,W), (S,L)

    Sequence: SYGKLSIFTMSWSL

  23. The sample was diluted 1000-fold. The concentration after dialysis is thus 0.001 M, or 1 mM. You could reduce the salt concentration by dialyzing your sample, now 1 mM, in more buffer free of (NH4)2SO4.