49. For a 0.1 M aqueous solution of lysine, the pH at which it carries no net charge is .

How to Calculate the Isoelectric Point (pI) of Lysine? | Complete CSIR NET Numerical Solution

Correct Answer

pI = 9.74

Introduction

Amino acids are amphoteric molecules because they contain both acidic and basic functional groups. Depending on the pH of the surrounding solution, an amino acid can exist as a positively charged ion, a negatively charged ion, or a zwitterion, in which the molecule carries both positive and negative charges but has no net electrical charge. The pH at which an amino acid exists predominantly in this electrically neutral form is known as its isoelectric point (pI). This property plays a crucial role in protein purification, electrophoresis, chromatography, and understanding protein solubility.

Lysine is classified as a basic amino acid because it possesses an additional ε-amino group in its side chain. Unlike neutral amino acids, lysine has three ionizable groups, making the calculation of its isoelectric point slightly different. For basic amino acids, the pI is obtained by averaging the two highest pKa values, which correspond to the two positively charged amino groups.

Understanding the Concept Behind the Question

Lysine contains three ionizable groups:

α-Carboxyl group (–COOH) with pKa ≈ 2.18
α-Amino group (–NH₃⁺) with pKa ≈ 8.95
ε-Amino side-chain group (–NH₃⁺) with pKa ≈ 10.53

At very low pH, all ionizable groups are protonated, giving lysine a net charge of +2.

As the pH increases:

The carboxyl group loses a proton first, reducing the net charge to +1.
The α-amino group then loses a proton, producing the zwitterionic form with a net charge of 0.
Finally, the side-chain amino group loses its proton, resulting in a net charge of –1.

The isoelectric point lies between the two pKa values that surround the neutral (zwitterionic) species. Therefore, for lysine, the pI is calculated using the second and third pKa values.

Step 1. Write the pKa Values

For lysine:

pKa₁ (–COOH) = 2.18

pKa₂ (α–NH₃⁺) = 8.95

pKa₃ (ε–NH₃⁺) = 10.53

Step 2. Identify the pKa Values Around the Zwitterion

The neutral species exists after the α-amino group loses its proton but before the side-chain amino group is deprotonated.

Therefore, the relevant pKa values are:

pKa₂ = 8.95

pKa₃ = 10.53

Step 3. Apply the Formula

For basic amino acids:

pI = (pKa₂ + pKa₃) / 2

Substituting the values:

pI = (8.95 + 10.53) / 2

pI = 19.48 / 2

pI = 9.74

Final Calculation

Isoelectric Point (pI) = 9.74

Why Is the Isoelectric Point Important?

The isoelectric point represents the pH at which an amino acid or protein has zero net electrical charge. At this pH, the molecule experiences minimal migration in an electric field and often exhibits its lowest solubility, making the pI extremely useful in protein purification techniques such as isoelectric focusing and selective precipitation.

For lysine, the high pI reflects its basic nature, as it contains two amino groups capable of accepting protons. Consequently, lysine remains positively charged over a broad pH range and becomes electrically neutral only under moderately alkaline conditions.

Ionization of Lysine with Increasing pH

The ionization of lysine can be summarized as follows:

Very Low pH

Carboxyl group protonated
Both amino groups protonated
Net charge = +2

↓

After loss of proton from –COOH

Carboxylate formed
Both amino groups remain protonated
Net charge = +1

↓

After loss of proton from α-amino group

One amino group neutral
Side-chain amino group still protonated
Net charge = 0 (Isoelectric Point)

↓

After loss of proton from ε-amino group

Both amino groups neutral
Carboxylate remains negative
Net charge = –1

Formula Summary

Neutral Amino Acids

pI = (pKa₁ + pKa₂) / 2

Acidic Amino Acids

pI = Average of the two lowest pKa values

Basic Amino Acids

pI = Average of the two highest pKa values

This rule is essential for solving amino acid pI problems in competitive examinations.

Biological Importance

The isoelectric point influences many biological and biochemical properties of proteins and amino acids, including solubility, electrophoretic mobility, enzyme activity, and molecular interactions. Proteins are least soluble at their pI because electrostatic repulsion between molecules is minimized, promoting aggregation and precipitation. This principle is widely used in biochemical purification methods and analytical techniques.

Lysine, being a basic amino acid with a high pI, contributes significantly to the positive charge of proteins, facilitating interactions with negatively charged molecules such as DNA, RNA, and phospholipid membranes.

High-Yield Points

Lysine is a basic amino acid.
Lysine has three ionizable groups.
For basic amino acids, calculate pI using the two highest pKa values.
Lysine pKa values:
- 2.18
- 8.95
- 10.53
pI of lysine = 9.74
At pI, the amino acid has zero net charge.

Frequently Asked Questions

Why are the first and second pKa values not averaged for lysine?

The neutral (zwitterionic) form of lysine exists between the second and third dissociation steps, so the pI must be calculated using pKa₂ and pKa₃ rather than pKa₁ and pKa₂.

Does amino acid concentration affect the isoelectric point?

No. The isoelectric point is an intrinsic property of the amino acid and depends only on its ionizable groups and their pKa values. Therefore, a 0.1 M solution has the same pI as any other concentration under identical conditions.

Why does lysine have a high pI?

Lysine possesses an additional ε-amino group that remains protonated until relatively high pH values, causing the molecule to remain positively charged over a wide pH range and giving it a high isoelectric point.

Key Takeaways

Lysine is a basic amino acid containing three ionizable groups. Because its neutral zwitterionic form exists between the dissociation of the α-amino group and the ε-amino side-chain group, its isoelectric point is calculated by averaging the two highest pKa values (8.95 and 10.53). Applying the formula:

pI = (8.95 + 10.53) / 2 = 9.74

Thus, lysine carries no net electrical charge at pH 9.74, regardless of whether the solution concentration is 0.1 M or any other concentration under physiological conditions.

Final Answer

Isoelectric Point (pI) = 9.74

Explanation

Lysine is a basic amino acid with three ionizable groups having pKa values of approximately 2.18, 8.95, and 10.53. Since the zwitterionic form lies between the deprotonation of the α-amino group and the ε-amino side-chain group, the isoelectric point is calculated by averaging the two highest pKa values: