Effect of pH on Protein Size Exclusion Chromatography Application Note Introduction Determination of protein structure is key to understanding the function of many proteins. Molecule size is an indicator of structure and provides information on the quaternary structure of the protein. Many proteins undergo profound conformational changes in different solution conditions, with the most extreme being denaturation. Similar structural modifications may occur as a function of ionic strength, pH, temperature and changes in various other solution conditions. Proteins comprise amino acids, some of which have polar side chains or contain acidic or basic groups. At the isoelectric point, the protein is uncharged and charge repulsions of similar functional groups will be at a minimum, allowing aggregation to take place. Many proteins precipitate under these conditions, and even for proteins that remain in solution at their isoelectric point this is the pH of minimum solubility. At physiological pH most proteins are above their isoelectric point and have a net negative charge. A pH lower than the isoelectric point causes protonation of acidic groups and a net positive charge on the protein. In each case, the like- charges formed repel each other, reducing the likelihood of protein aggregation. However, in areas of larger charge density the intramolecular repulsion can lead to destabilization of the protein tertiary structure and subsequent unfolding. In some cases, this unfolding may lea...
This document is an application note from Agilent Technologies regarding the impact of pH on protein size exclusion chromatography. It explains how proteins undergo conformational changes and aggregation based on solution conditions like pH, ionic strength, and temperature. The note specifically investigates how varying pH levels affect the retention behavior and elution profiles of globular proteins.
| Column | ProSEC 300S, 300 x 7.5 mm |
|---|---|
| Eluent | 0.1 M KH2PO4 containing 0.3 M NaCl |
| Flow Rate | 1.0 mL/min |
| Injection Volume | 100 mL |
| Temperature | 5°C |
| Detection | UV at 310 nm |
ProSEC 300S columns are not designed for continual use above pH 7.5.
Reducing the pH to 4.0 results in less defined peaks and an increase in protein-matrix interactions, partially retaining the protein on the column.