Protein Isolation Practice Problems (biochem)

You are using ammonium sulfate to purify protein Q (pI = 5.0) by

salting out from a solution at pH 7.0. How should you adjust the pH of

the mixture to maximize the amount of protein Q that precipitates?

lowering pH from 7.0 to 5.0

(a) In what order would the amino acids Arg, His, and Leu be eluted

from a carboxymethyl column at pH 6? (b) In what order would Glu,

Lys, and Val be eluted from a diethylaminoethyl column at pH 8?

a) Leu, His, Arg

b)Lys, Val, Glu

Explain how you could use a column containing carboxymethyl

groups to separate serum albumin and ribonuclease A

At neutral pH(7), serum albumin (pI 4.9) is negatively charged, and ribonuclease A (pI 9.4) is positively charged. Serum albumin will therefore flow through a cation exchange column and can be recovered, while ribonuclease A will interact with the carboxymethyl groups and can be recovered later by increasing the salt concentration or increasing the pH

Explain why a certain protein has an apparent molecular mass of

90 kD when determined by gel fi ltration and 60 kD when determined

by SDS-PAGE in the presence or absence of 2-mercaptoethanol.

Which molecular mass determination is more accurate?

The protein behaves like a larger protein during gel filtration, suggesting that it has an elongated shape. The mass determined by SDS-PAGE is more accurate since the mobility of a denatured SDS-coated protein depends only on its size.

Determine the subunit composition of a protein from the following

information:

Molecular mass by gel filtration: 200 kD

Molecular mass by SDS-PAGE: 100 kD

Molecular mass by SDS-PAGE with 2-mercaptoethanol:

40 kD and 60 kD

The protein contains two 60-kD polypeptides and two 40-kD

polypeptides. Each 40-kD chain is disulfide bonded to a 60-kD

chain. The 100-kD units associate noncovalently to form a protein with a molecular mass of 200 kD

A protein has an apparent mass of 800 kD by gel fi ltration chro-

matography, but SDS-PAGE shows a single band at a position cor-

responding to 200 kD. In an ultracentrifuge, will the protein exhibit a

sedimentation coefficient corresponding to 200 kD or 800 kD?

The protein appears to be a tetramer of 200-kD subunits. The subunits separate under the denaturing conditions of SDS-PAGE (giving an apparent mass of 200 kD). Both gel filtration and ultra-centrifugation are performed under nondenaturing conditions, so the protein behaves as an 800-kD particle

Explain how you would use salting out to prepare a more concen-

trated solution of a protein.

Add a salt to dilute protein solution to reach a final salt concentration that is somewhat higher than the salting-out point. Centrifuge the solution, collect the protein precipitate, and then dissolve in the precipitate in a small volume of buffer

The salting out procedure outlined in Fig. 5-5 results in significant protein purification by eliminating many less- and more-soluble proteins. What is its other advantage?

Following salting out, the precipitated protein can be redissolved in a very small volume,

thus concentrating it. A highly concentrated protein solution may be necessary for a

subsequent gel filtration step or other analytical procedure. Concentrated protein solutions

are also easier to handle, and they minimize protein loss by adsorption to surfaces.

The pI of pepsin is < 1.0, whereas that of lysozyme is 11.0. What amino acids must predominate in each protein to generate such pI's?

pepsin, aspartate and glutamate must predominate because they have acidic R groups.

Lysine, arginine, and tyrosine, all with basic R groups, must predominate in lysozyme

Evaluate the solubility of the following peptides at the given pH. For each pair, which peptide is more soluble and why? Hint: Evaluate the ionization states of the amino acid functional groups.

(a) pH 7: Gly20 and (Glu-Asp)10

(b) pH 5: (Cys-Ser-Ala)5 and (Pro-Ile-Leu)5

(c) pH 7: Leu3 and Leu10

(a) At pH 7.0, the side chains of Asp and Glu are ionized. Although both peptides contain

ionized N- and C-terminal groups, (Glu-Asp)10 contains more onized groups than Gly20

and is therefore the more soluble peptide.

(b) Although Cys would be ionized at higher pH, neither peptide has ionized R groups at

pH 5.0. However, (Cys-Ser-Ala)5 has more polar groups and is therefore more soluble

than (Pro-Ile-Leu)5.

(c) Leu3 is less hydrophobic since it has two charged groups (its N- and C-termini) per

three residues, compared to two charged groups per 10 residues in Leu10. The shorter

peptide is more soluble since it requires less disruption of water structure to dissolve.

Would you use an anion or cation exchange column to purify bovine histone at pH 7.0?

Bovine histone is highly basic (pI = 10.8) and therefore positively charged at pH 7.0. It would therefore bind to the anionic matrix of a cation exchanger (e.g., carboxymethyl cellulose).

Why is a DEAE ion exchange column ineffective above pH 9?

Above pH 9, the amino groups of the DEAE matrix lose their protons and are therefore unable to bind anionic substances.

You have a protein whose molecular mass is about 6.8 kD according to SDS-PAGE.

However, SDS-PAGE of the protein in the presence of 2-mercaptoethanol reveals two

bands of 2.3 kD and 4.5 kD. Are these polypeptides too large for direct sequencing by an

automated sequencer using Edman degradation? What steps must you take to determine the

complete sequence of this protein?

Since the average mass of an amino acid residue is ~110 D, a peptide with a mass of 4500

D contains ~44 residues. Thus, both peptides are small enough to be directly sequenced.

The two peptides are linked by a disulfide bond, so determining the protein's complete

sequence requires the following steps: (a) reduce the disulfide bond; (b) alkylate the free

thiol groups; (c) separate the peptides; and (d) sequence each peptide. If either peptide

contains more than one Cys residue, additional fragmentation and sequencing steps would

be necessary to identify the position of the disulfide bond in the intact protein.