SEATWORK #2

Section: BSN1J
Group No. : Group 1
Group Leader: JACILDO, Kuh Kyla C. Group Members:
Destua, Polen Love V.
Lawaan, Renalyn
Monzon, Samantha Kate H.
Olicia, Diana Rose
Patao, Anneliese Sheri M.

a. From the information given in the table, calculate the specific activity of the enzyme after
each purification procedure.
- After step 1, it is 200 units/mg. Step 2 is 600 units/gm; step 3 is 250 units/mg;
step 4 is 4,000 units/mg; step 5 is 1500 units/mg; and step 6 is 1500 units/mg.
b. Which of the purification procedures used for this enzyme is most effective (ie., gives the
greatest relative increase in purity)?
- Step 4 which is the ion-exchange chromatography.
c. Which of the purification procedures is least effective?
 - The purification procedure that is least effective is Step 3, which is the
precipitation (pH).
d. Is there any indication based on the results shown in the table that the enzyme after step 6
is now pure?
e. What else could be done to estimate the purity of the enzyme preparation?
- Yes, specific activity did not increase in step 6.Protein can be purified by mass
using the electrophoresis process known as SDS-PAGE. Could be applied to
additional assessments.

2. Mixtures of amino acids can be analyzed by first separating the mixture into its
components through ion-exchange chromatography. Amino acids placed on a
cation-exchange resin containing sulfonate (–SO3-) groups flow down the column at
different rates because of two factors that influence their movement: (1) ionic attraction
between the sulfonate residues on the column and positively charged functional groups on
the amino acids, and (2) hydrophobic interactions between amino acid side chains and the
strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids
listed, determine which will be eluted first from the cation-exchange column by a pH 7.0
buffer. (a) Asp and Ly,(b) Arg and Met, (c) Glu and Val, (d) Gly and Leu, (e) Ser and Ala

(a) Asp and Ly: Aspartic acid (Asp) has a carboxyl group that can be negatively charged at pH
7.0, while lysine (Ly) has an amino group that can be positively charged at pH 7.0. Therefore, Ly
will be retained by the cation-exchange column and eluted first, while Asp will be eluted later.

(b) Arg and Met: Arginine (Arg) has multiple positive charges at pH 7.0 due to its guanidinium
group, while methionine (Met) does not have any charged functional groups at pH 7.0.
Therefore, Met will be eluted first, while Arg will be retained by the cation-exchange column
and eluted later.

(c) Glu and Val: Glutamic acid (Glu) has a carboxyl group that can be negatively charged at pH
7.0, while valine (Val) does not have any charged functional groups at pH 7.0. Therefore, Val
will be eluted first, while Glu will be retained by the cation-exchange column and eluted later.
(d) Gly and Leu: Glycine (Gly) and leucine (Leu) do not have any charged functional groups at
pH 7.0. In this case, the hydrophobic interactions between the amino acid side chains and the
hydrophobic backbone of the polystyrene resin will be the main factor influencing their
movement. However, it is not possible to determine which amino acid will elute first based
solely on this information.

(e) Ser and Ala: Serine (Ser) has a hydroxyl group that can be negatively charged at pH 7.0,
while alanine (Ala) does not have any charged functional groups at pH 7.0. Therefore, Ala will
be eluted first, while Ser will be retained by the cation-exchange column and eluted later.

3. A peptide has the sequence Glu–His–Trp–Ser–Gly–Leu–Arg–Pro–Gly (a) What is the net

charge of the molecule at pH 3, 8, and 11? (Use pKa values for side chains and terminal
amino and carboxyl groups as given in lab activity 2.) (b) Estimate the pI for this peptide.

a. The terminal amine, carboxylic acid, and the ionizable side chains present in the
molecule all affect the net charge of the peptide chain. The only monomers having
ionizable side chains are Glu, His, and Arg, while the N- and C-terminal amino groups
are Glu and Gly, respectively. We take into account the Pka given below.

𝑝𝐾𝑎1 𝑝𝐾𝑎2 𝑝𝐾𝑎3

Glu 2.19 9.67 4.25

His 1.82 9.17 6.00

Arg 2.17 9.04 12.48

Gly 2.34 9.60 —

pH 3
At this very acidic pH, we expect that aloof the functional groups in the peptide are protonated,
except for the carboxyl group of the C-terminal amino acid, Gly (𝑝𝐾𝑎12.34 < pH 3)

Functional groups with positive charges are in red, while groups with negative charge are in blue.
Net charge= (+1) + (+1) + (+1) + (-1) = +2
● The net charge of the peptide at pH 3 is +2
pH 8
At pH 8, those groups with 𝑝𝐾𝑎𝑠 < 8 become deprotonated: the carboxyl group of Gly , side

chain od His, and side chain of Glu.

● At pH 8, the net charge of the peptide is 0

pH 11
At pH 11, all ionizable functional groups are deprotonated, except for the side chain of Arg (
𝑝𝐾𝑎3 12.48 > pH 11)

Net charge = (-1) + (+1) + (-1) = -1

● At pH 11, the net charge of the peptide is -1

4. What functional groups must be present to confer this pI on pepsin? The quantity of
negatively charged groups in proteins must be high. Those molecules must have
carboxylate groups.Which aminoacids in the proteins would contribute to such groups?
Amino acids such as aspartic acid or aspartate (Asp) and glutamic acid or glutamate (Glu)
would contribute to such groups.

- Pepsin is the name given to a mix of several digestive enzymes secreted (as larger
precursor proteins) by glands that line the stomach. These glands also secrete
hydrochloric acid, which dissolves the particulate matter in food, allowing pepsin to
enzymatically cleave individual protein molecules. The resulting mixture of food, HCl,
and digestive enzymes is known as chyme and has a pH near 1.5. What pI would you
predict for the pepsin proteins? For the pepsin to be soluble, the pI value should be close
to the pH of gastric juice, or pI 1.

5. At he protein allergen from peanuts, a protein called Ara h8 was recently purified and
characterized. The investigators initially had difficulty separating Ara h8 from a similar
protein in peanuts called Ara ho because the two proteins were of similar size and had
nearly identical pl values. Separation of the two proteins was finally achieved when it was
noted that the Ara ho protein contained ten cysteine residues involved in disulfide bridges.
whereas Ara h8 contained no cysteines. The protein mixture was treated with a reducing
agent, dithiothreitol (DTT), and then treated with iodoacetic acid (ICH COOH, a reagent
that adds to, or alkylates, an -SH group and releases free iodine). The mixture was then
loaded onto an anion exchange column and the two proteins were successfully separated.

a. When Cys residues are exposed to DTT followed by iodoacetic acid, the following
structural changes occur:
1. DTT (dithiothreitol) is a reducing agent that breaks the disulfide bonds between cysteine
residues. It converts the cysteine residues (-SH) into thiol groups (-SH2).
2. Iodoacetic acid (ICH COOH) is an alkylating agent that reacts with the thiol groups
(-SH2) to form stable thioether bonds. This reaction involves the addition of iodoacetic
acid to the thiol group, releasing free iodine.

So, the structural changes can be represented as follows:

Cysteine residues before treatment: -S-S-

After treatment with DTT: -SH2 + -SH2

After treatment with iodoacetic acid: -S-CH2-COOH + I2

b. The plausible elution profile for the separation of the proteins by anion exchange
chromatography would typically show the protein concentration versus solvent volume. The
profile would typically have the following shape:

- Initially, the proteins are loaded onto the column and are not yet separated. The
concentration of the protein mixture is high at this point.
- As the solvent is passed through the column, the proteins start to interact with the anion
exchange resin. The protein with no cysteine residues (Ara h8) will have a lower affinity
for the resin compared to the protein with cysteine residues (Ara ho). Thus, Ara h8 will
elute first, followed by Ara ho.
- The concentration of Ara h8 in the eluate will initially increase and then gradually
decrease as more of the protein is eluted.
- The concentration of Ara ho in the eluate will initially be low but will gradually increase
as more of the protein is eluted.

c. This treatment resulted in the successful separation of the proteins because:

 - DTT reduced the disulfide bonds present in Ara ho, converting them into thiol groups.
This change in structure reduced the affinity of Ara ho for the anion exchange resin.
- Iodoacetic acid then reacted with the thiol groups in Ara ho, forming stable thioether
bonds. This further altered the structure of Ara ho and decreased its affinity for the resin.
- As a result, Ara ho, with its altered structure and reduced affinity for the resin, eluted
later than Ara h8 during the anion exchange chromatography. This allowed for the
successful separation of the two proteins.

6. A quantitative amino acid analysis reveals that bovine serum albumin (BSA) contains
0.58% tryptophan (MW 204) by weight. (a) Calculate the minimum molecular weight of
BSA (i.c., assume there is only one Trp residue per protein molecule). (b) Gel filtration of
BSA gives a molecular weight estimate of 70,000. How many Trp residues are present in a
molecule of serum albumin?

a. To calculate the minimum molecular weight of BSA, we need to use the percentage of
tryptophan and its molecular weight.

Given that BSA contains 0.58% tryptophan by weight, we can calculate the minimum molecular
weight of BSA as follows:

Percentage of tryptophan = (Mass of tryptophan / Mass of BSA) * 100

0.58 = (204 / Mass of BSA) * 100

Solving for the mass of BSA:

Mass of BSA = 204 / (0.58/100) = 35,172.41 g/mol

Therefore, the minimum molecular weight of BSA is approximately 35,172.41 g/mol.

b. Gel filtration provides an estimate of the molecular weight of a protein based on its elution
volume compared to the elution volumes of standard proteins of known molecular weights.

Given that the molecular weight estimate of BSA from gel filtration is 70,000, we can calculate
the number of tryptophan residues present in a molecule of serum albumin as follows
Number of Trp residues = (Molecular weight of BSA) / (Molecular weight of tryptophan)

Number of Trp residues = 70,000 / 204 ≈ 343

7. A protein has a molecular mass of 400 kDa when measured by gel filtration. When
subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), the protein
gives three bands with molecular masses of 180, 160, and 60 kDa. When electrophoresis is
carried out in the presence of SDS and dithiothreitol, three bands are again formed, this
time with molecular masses of 160, 90, and 60 kDa. Determine the subunit composition of
the protein.

In gel electrophoresis, the SDS agent produces denaturation of the protein and confers negative
charge, so the protein subunits can migrate according to their masses. It produces dissociation of
the protein in its subunits but it cannot disrupt disulphyde bridges (S-S) that can bond subunits
together.

From the data, with SDS we observe 3 bands → 180 kDa + 160 kDa + 60 kDa

The addition of dithiotreitol (DTT), a reducing agent, produces the disruption of disulphyde
bridges. From the data:

With DTT → 160 kDa + 90 kDa + 60 kDa

We observe that 160 kDa and 60 kDa subunits are conserved (they are the same as with SDS
only), but 180 kDa subunit is missing and in its place appears a band of 90 kDa - a half 180 kDa.

So, the band at 180 kDa is composed by two subunits bonded by a disulphyde bridge.

Therefore, the composition of the protein is: 1 subunit of 160 kDa, 2 subunits of 90 kDa and

1 subunit of 60 kDa.