The theoretical pI (isoelectric point) is the pH at which a protein has no net electrical charge, a balance between positive and negative charges. According to the reference, it's calculated based on the primary sequence of the protein. However, the reference notes that the theoretical pI is "unlikely to match the actual pI because some of the charged side chains are either buried or in salt bridges."
Here's a breakdown:
- Definition: The pH value where the sum of positive charges equals the sum of negative charges on a protein.
- Calculation: Determined from the amino acid sequence. The pKa values of ionizable groups (e.g., amino and carboxyl groups of amino acids, and the N- and C-termini) are used in the calculation.
- Discrepancy: The theoretical pI often differs from the experimentally determined pI.
Why the Difference?
The theoretical pI is a prediction based purely on the amino acid sequence. Several factors can cause the actual, experimentally-determined pI to differ from the theoretical value:
- Buried Charges: Some charged amino acid side chains may be buried within the protein structure, shielded from the solvent and therefore not contributing to the overall charge.
- Salt Bridges: Interactions between oppositely charged amino acids (salt bridges) can neutralize charges.
- Post-Translational Modifications: Modifications like glycosylation, phosphorylation, or acetylation can add or remove charges, shifting the pI.
- Buffer Conditions: The specific buffer used during experimental determination can influence the observed pI.
Importance of pI
Understanding the pI of a protein is crucial for several reasons:
- Protein Purification: pI is used in techniques like isoelectric focusing to separate proteins based on their charge.
- Protein Stability: Proteins are often least soluble and most prone to aggregation at their pI.
- Enzyme Activity: The activity of some enzymes is pH-dependent and can be affected by proximity to the protein's pI.
