Q.43 The C-terminal carboxyl group and the N-terminal amino group in amino acids have a dissociation
constant ( 𝐩𝐊𝐚
) of 2.2 and 9.2 , respectively. The 𝐩𝐊𝐚 of side chain carboxyl group in glutamic acid is 4.2
and side chain amino group in lysine is 10.2 . The difference in isoelectric point (pI) of lysine and glutamic
acid (rounded off to two decimal places) is
____ .

Understanding Isoelectric Point (pI) in Amino Acids: Lysine vs. Glutamic Acid Difference Calculation

The isoelectric point (pI) is the pH at which an amino acid has no net charge, crucial for protein separation techniques like electrophoresis. Lysine, a basic amino acid, and glutamic acid, an acidic one, differ due to their side chains, leading to a pI difference of 5.50 when calculated using given pKa values.

pI Calculation Method

For amino acids with ionizable side chains, pI is the average of pKa values flanking the zero-charge species. All amino acids share C-terminal pKa (2.2) and N-terminal pKa (9.2); glutamic acid adds side chain carboxyl pKa (4.2), lysine adds side chain amino pKa (10.2).​

• Acidic amino acids (extra carboxyl): pI = (pKa1 + pKa_side chain)/2, where pKa1 is the lowest (C-terminal, 2.2).

• Basic amino acids (extra amino): pI = (pKa2 + pKa_side chain)/2, where pKa2 is the N-terminal (9.2).

This averages the two pKa values where the predominant form shifts from +1 to 0 (acidic) or 0 to -1 (basic).

Glutamic Acid pI Step-by-Step

Glutamic acid has three pKa: 2.2 (C-terminal COOH), 4.2 (side chain COOH), 9.2 (N-terminal NH3+). The zero net charge form (COO-, COO-, NH3+) exists between pKa1 (2.2) and pKa2 (4.2).​

Thus, pI = (2.2 + 4.2)/2 = 6.4/2 = 3.20.

Lysine pI Step-by-Step

Lysine has three pKa: 2.2 (C-terminal COOH), 9.2 (N-terminal NH3+), 10.2 (side chain NH3+). The zero net charge form (COO-, NH3+, NH3+) exists between pKa2 (9.2) and pKa3 (10.2).​

Thus, pI = (9.2 + 10.2)/2 = 19.4/2 = 9.70.

Correct Answer and Difference

The pI difference is |9.70 – 3.20| = 6.50 (rounded to two decimal places). This matches exam solutions where glutamic acid pI ≈ 3.2 and lysine pI ≈ 9.7.​