Proteomics/Protein Separations - Chromatography/Cation Exchangers
Cation Exchangers
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Cation exchangers are named for their ability to attract cations or positively charged particles. In this case, the resin of the chromatography system is negatively charged and proteins will bind if the buffer pH is less than the protein’s unique isoelcetric point.
Just as with anion exchangers, cation exchangers can be classified as either weak or strong. A weak cation exchanger is comprised of a weak acid that gradually loses its charge as the pH decreases, while a strong anion exchanger is comprised of a strong acid that is able to sustain its charge over a wide pH range. Carboxymethal groups are used commonly as weak cation exchangers, while sulfopropyl groups are widely used as strong cation exchangers (Res1)
Designing a Cation Exchange Separation
[edit | edit source]Selecting the correct resin and buffer is critical as it determines the binding properties of proteins to the resin. In cation exchange, the protein of interests needs to be positively charge to bind to the negatively charged stationary phase. Suppose we have a mixture of the following protein :
| Protein | pI | pH 4.8 cm | pH 7.2 cm | pH 8 cm |
| Carbonic Anhydrase | 7.0 | +16.5 | -0.4 | -2.7 |
| Carboxypeptidase B | 6.2 | +12.0 | -3.3 | -6.3 |
| Chymotrypsin | 8.0 | +9.0 | +2.7 | 0.0 |
| Lysozyme | 9.8 | +14.1 | +7.9 | +6.9 |
- At pH 4.8 cm all protein will bind to the cation exchanger, with carbonic anhydrase binding the strongest to the stationary. If elution is performed at this pH, the protein will elute in the order of chymotrypsin, carboxypeptidase B, lysozyme, and carbonic anhydrase.
- At pH 7.2 cm, cm, carbonic anhydrase and carboxypeptidase B will elute in the wash (before the gradient is initiated), followed by chymotrypsin, then lysozyme during the salt gradient.
- AtpH 8 cm, only the lysozyme will bind to the stationary phase, the other three proteins will elute in the wash.
It is also possible to refine the salt concentration in solvents A and B so that the more weakly bound proteins will wash off the column in the wash, even if they are positively charged. For example, if you modify pH 4.8 cm to have 0.1 M NaCl in solvent A and 0.2 M NaCl in solvent B, you find that the most weakly bound protein (chymotrypsin) elutes in the wash, while the other three elute in the gradient.
Resources
[edit | edit source]- Craig. P, Rochester Institute of Technology, Designing a Cation Exchange Separation