Question

In paper electrophoresis, amino acids and peptides can be separated by their differential migration in an electric field. To the center of a strip of paper, wet with buffer at pH 6, is applied a mixture of the following three peptides in a single small spot: Gly-Lys, Gly-Asp, and Gly-Ala. A positively charged electrode (anode) is attached to the left side of the paper, and a negatively charged electrode (cathode) to the right side. A voltage is applied across the ends of the paper for a time, after which the peptides have separated into three spots: one near the cathode, one near the anode and one in the center, at the location of the original spot. Which statement is incorrect?

• A. Gly-Lys migrates to the cathode
• B. Gly-Asp migrates to the anode
• C. Gly-Ala does not migrate
• D. Gly-Lys is a acidic dipeptide

Answer 1

Answer: (D)

Gly-Lys is a acidic dipeptide

Answer 2

Paper electrophoresis separates charged molecules based on their differential migration in an electric field. The direction and speed of migration depend on the net charge of the molecule, the strength of the electric field, and the properties of the support medium and buffer. At a given pH, a molecule with a net positive charge will migrate towards the cathode (negative electrode), a molecule with a net negative charge will migrate towards the anode (positive electrode), and a molecule with a net zero charge (at its isoelectric point, pI) will not migrate.

We need to determine the net charge of each peptide at pH 6. The ionizable groups in a peptide are the N-terminal amino group, the C-terminal carboxyl group, and any ionizable side chains. We use approximate pKa values for these groups:

• N-terminal amino group: pKa ≈ 8.0
• C-terminal carboxyl group: pKa ≈ 3.1
• Lysine side chain amino group: pKa ≈ 10.5
• Aspartic acid side chain carboxyl group: pKa ≈ 3.9

At pH 6:

• If pH < pKa, the group is predominantly protonated.
• If pH > pKa, the group is predominantly deprotonated.

1. Gly-Lys:

   • N-terminal amino group (pKa ≈ 8.0): pH 6 < 8.0, so it is protonated (-NH$_3^+$), charge +1.
   • C-terminal carboxyl group (pKa ≈ 3.1): pH 6 > 3.1, so it is deprotonated (-COO$^-$), charge -1.
   • Lysine side chain amino group (pKa ≈ 10.5): pH 6 < 10.5, so it is protonated (-NH$_3^+$), charge +1.

   Net charge of Gly-Lys at pH 6 = (+1) + (-1) + (+1) = +1. Since Gly-Lys has a net positive charge, it will migrate towards the cathode (negative electrode).

2. Gly-Asp:

   • N-terminal amino group (pKa ≈ 8.0): pH 6 < 8.0, so it is protonated (-NH$_3^+$), charge +1.
   • C-terminal carboxyl group (pKa ≈ 3.1): pH 6 > 3.1, so it is deprotonated (-COO$^-$), charge -1.
   • Aspartic acid side chain carboxyl group (pKa ≈ 3.9): pH 6 > 3.9, so it is deprotonated (-COO$^-$), charge -1.

   Net charge of Gly-Asp at pH 6 = (+1) + (-1) + (-1) = -1. Since Gly-Asp has a net negative charge, it will migrate towards the anode (positive electrode).

3. Gly-Ala:

   • N-terminal amino group (pKa ≈ 8.0): pH 6 < 8.0, so it is protonated (-NH$_3^+$), charge +1.
   • C-terminal carboxyl group (pKa ≈ 3.1): pH 6 > 3.1, so it is deprotonated (-COO$^-$), charge -1.
   • Alanine side chain has no ionizable group.

   Net charge of Gly-Ala at pH 6 = (+1) + (-1) = 0. Since Gly-Ala has a net zero charge, it will not migrate and will remain at the origin.

The electrophoresis setup has the anode on the left and the cathode on the right.

• Gly-Lys (charge +1) migrates towards the cathode (right).
• Gly-Asp (charge -1) migrates towards the anode (left).
• Gly-Ala (charge 0) remains at the center.

This matches the observed separation into three spots: one near the cathode, one near the anode, and one in the center.

Now let's evaluate the given statements:

1. Gly-Lys migrates to the cathode: Our analysis shows Gly-Lys has a net charge of +1 at pH 6 and migrates to the cathode. This statement is correct.
2. Gly-Asp migrates to the anode: Our analysis shows Gly-Asp has a net charge of -1 at pH 6 and migrates to the anode. This statement is correct.
3. Gly-Ala does not migrate: Our analysis shows Gly-Ala has a net charge of 0 at pH 6 and does not migrate. This statement is correct.
4. Gly-Lys is a acidic dipeptide: Gly-Lys contains a Lysine residue, which has a basic side chain (pKa ≈ 10.5). At pH 6, the Lysine side chain is protonated (+1). The overall net charge of Gly-Lys at pH 6 is +1. Peptides containing basic amino acids like Lysine or Arginine are considered basic peptides. Acidic peptides contain acidic amino acids like Aspartic acid or Glutamic acid. Therefore, Gly-Lys is a basic dipeptide, not an acidic one. This statement is incorrect.

The question asks for the incorrect statement. The fourth statement is incorrect.