Question

Do the following compounds migrate to the cathode or to the anode on electrophoresis at the specified \(\mathrm{pH}\) ? (a) Histidine at pH \(6.8\) (b) Lysine at \(\mathrm{pH} 6.8\) (c) Glutamic acid at \(\mathrm{pH} \mathrm{} 4.0\) (d) Glutamine at \(\mathrm{pH} 4.0\) (e) Glu-Ile-Val at pH \(6.0\) (f) Lys-Gln-Tyr at pH \(6.0\)

Short answer

(a) Histidine at pH 6.8: Answer: Cathode (b) Lysine at pH 6.8: Answer: Anode (c) Glutamic acid at pH 4.0: Answer: Cathode (d) Glutamine at pH 4.0: Answer: Anode (e) Glu-Ile-Val at pH 6.0: Answer: Cathode (f) Lys-Gln-Tyr at pH 6.0: Answer: Anode

Step-by-step solution

1. Determine the pKa and pI values for each amino acid

The pKa values can be found in biochemistry references, textbooks, or online resources. The pI (isoelectric point) can be calculated by averaging the pKa values of the relevant ionizable groups. (a) Histidine: - pKa values: \(\alpha\)-COOH (\(1.8\)), \(\alpha\)-NH₃⁺ (\(9.3\)), imidazole side chain (\(6.0\)) - pI: \( (1.8 + 9.3) / 2 = 5.6\) (b) Lysine: - pKa values: \(\alpha\)-COOH (\(2.2\)), \(\alpha\)-NH₃⁺ (\(9.0\)), \(\epsilon\)-NH₃⁺ (\(10.5\)) - pI: \( (9.0 + 10.5) / 2 = 9.75\) (c) Glutamic acid: - pKa values: \(\alpha\)-COOH (\(2.2\)), \(\alpha\)-NH₃⁺ (\(9.7\)), \(\beta\)-COOH (\(4.3\)) - pI: \( (2.2 + 4.3) / 2 = 3.25\) (d) Glutamine: - pKa values: \(\alpha\)-COOH (\(2.2\)), \(\alpha\)-NH₃⁺ (\(9.1\)) - pI: \( (2.2 + 9.1) / 2 = 5.65\) (e) Glu-Ile-Val (tripeptide): - Glu: pKa values of side chain: \(\beta\)-COOH (\(4.3\)) - Ile and Val: nonpolar, no ionizable groups - pI: Since the side chain of Glu has a COOH group, the peptide's pI is close to \(4.3\). (f) Lys-Gln-Tyr (tripeptide): - Lys: pKa values of side chain: \(\epsilon\)-NH₃⁺ (\(10.5\)) - Gln: no ionizable side chain - Tyr: pKa value of side chain: phenol group (\(10.5\)) - pI: Since the side chains of Lys and Tyr have ionizable groups, the peptide's pI is \((10.5 + 10.5)/2=10.5\).

2. Compare the pH of the solution to the pI of the amino acid to determine charge.

If the pH is greater than the pI then the compound will have a net negative charge, while if the pH is less than the pI, the compound will have a net positive charge. (a) Histidine at pH \(6.8\): - pH \(6.8\) \(>\) pI \(5.6\) \(\Rightarrow\) net negative charge - Migrates to: Cathode (b) Lysine at pH \(6.8\): - pH \(6.8\) \(<\) pI \(9.75\) \(\Rightarrow\) net positive charge - Migrates to: Anode (c) Glutamic acid at pH \(4.0\): - pH \(4.0\) \(>\) pI \(3.25\) \(\Rightarrow\) net negative charge - Migrates to: Cathode (d) Glutamine at pH \(4.0\): - pH \(4.0\) \(<\) pI \(5.65\) \(\Rightarrow\) net positive charge - Migrates to: Anode (e) Glu-Ile-Val at pH \(6.0\): - pH \(6.0\) \(>\) pI \(\approx 4.3\) \(\Rightarrow\) net negative charge - Migrates to: Cathode (f) Lys-Gln-Tyr at pH \(6.0\): - pH \(6.0\) \(<\) pI \(10.5\) \(\Rightarrow\) net positive charge - Migrates to: Anode

Key concepts

Isoelectric Point (pI)

The isoelectric point, or pI, of an amino acid is a crucial concept in understanding its behavior during electrophoresis. The pI is the pH value at which an amino acid has no net charge. At this point, there's an equal amount of positively and negatively charged species of the molecule. Understanding an amino's acid pI is key as it influences how it will migrate in an electric field, which is dependent on its charge.

To determine an amino acid's pI, we look at the ionizable groups in its structure. Most amino acids have at least two pKa values, one for the carboxyl group and one for the amino group. Some amino acids also have ionizable side chains. By averaging the pKa values of the ionizable groups that lose or gain protons near the pI, we arrive at the isoelectric point. This is an average of the pKa values for groups that can be both positively and negatively charged, such as the carboxyl and amino groups.

Amino Acid Charge at pH

The charge of an amino acid at a given pH level is another vital concept in predicting how it will migrate during electrophoresis. If the solution's pH is lower than the amino acid's pI, the amino acid will have a net positive charge since more protons are available to bind to the molecule. On the other hand, if the pH is higher than the pI, the amino acid will have a net negative charge as protons will dissociate.

For example, an amino acid with a pI of 6 will have a net positive charge in a pH 5 environment and a net negative charge at pH 7. This property becomes particularly important when we examine amino acids, or even polypeptides, in different pH environments, and it dictates whether they will move towards the cathode (negative electrode) or the anode (positive electrode) in an electric field.

Amino Acid Migration Direction

In electrophoresis, the migration direction of an amino acid is directly related to its charge, which, as we discussed, depends on the surrounding pH in relation to its pI. If an amino acid has a net positive charge, it will migrate towards the cathode (negative electrode), whereas with a net negative charge, it migrates towards the anode (positive electrode).

This principle allows us to separate a mixture of amino acids based on their pIs and the pH of the medium. In practical applications, such as protein purification or characterization, knowing the migration direction can offer insights into the amino acids' or peptides' structure and charge properties, facilitating better understanding and manipulation in biochemical studies.

Calculating pKa Values

Calculating pKa values is a fundamental skill in biochemistry that involves understanding the dissociation of acids. pKa is the logarithmic measure of the acid dissociation constant (Ka) and indicates the strength of an acid in solution. The lower the pKa value, the stronger the acid (more readily it donates protons).

For amino acids, we often use pKa values to predict charging properties and behavior in different pH environments. These values are typically determined experimentally; however, they can also be found in various reference materials. When we know the pKa values of the functional groups within an amino acid, we can calculate the pI, as seen in the step-by-step solution provided, and then predict the charge at different pH levels.