Molecular Techniques and Methods

Protein Purification by Ion-Exchange HPLC

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

  • Ion-exchange high-performance liquid chromatography (HPLC) separates proteins based on molecular charge. Protein mixtures are applied to an oppositely charged, chromatographic matrix, and the various proteins are bound by reversible, electrostatic interactions. The adsorbed proteins are eluted in order of least to most strongly bound molecules by increasing the ionic strength of the elution buffer, collected as individual chromatographic fractions, and analyzed separately.

  • Ion-exchange HPLC differs from reversed-phase (RP) HPLC in that the separation mechanism involves an ionic rather than a hydrophobic interaction between the protein and the chromatographic support.

  • Salt gradients are used to increase the ionic strength of the mobile phase and to reduce the strength of electrostatic interactions. They also act selectively to displace the charged residues of the retained proteins from ionic sites on the support surface. The displacing power of salts varies, with divalent salts being generally stronger than monovalent species.

  • In addition to salt gradients, pH gradients can be used. Protein charge can be affected by small changes in pH (e.g., decreases in solution pH will lower the negative charge, and reciprocally, a pH increase will reduce the positive charge). Negatively charged proteins can be eluted from an anion-exchange chromatography column with a decreasing pH gradient, and positively charged proteins can be eluted from a cation-exchange chromatography column with an increasing pH gradient.

  • Because linear pH gradients cannot be readily produced, salt gradients have become the de facto standard for protein elution in ion-exchange chromatography.



    Choice of HPLC Column
    If the pI (isoelectric point) of a protein is < 7, then anion-exchange chromatography should be used. If the pI of a protein is > 7, then cation-exchange chromatography should be used. In anion exchange, proteins containing a higher ratio of acidic to basic charges will be retained by the positively charged amines on the support surface. Conversely, in cation exchange, proteins with an excess of basic charges will be attracted to the negatively charged carboxyl (or other) acidic groups. Most ion-exchange columns have shorter lifetimes than reversed-phase columns. Since 5 cm, 10 cm, 15 cm, or 25 cm columns give similar resolution, the lower costs of the shorter columns is economical. Hence, shorter columns should generally be used. The loading capacity of a 4.6 mm i.d. x 25 cm HPLC column is 10 mg of protein.

    HPLC Column Handling
    Columns should be stored in 50% methanol after flushing out buffer salts with water. Never allow a column to dry out. Increasing column backpressure usually indicates a blockage in the column inlet frit. The column should be disconnected from the detector, reversed, and flushed with a gradient. Alternatively, the column may be disassembled, and the column inlet frit or the fitting with a pressed frit may be sonicated in 20% nitric acid followed by water or just replaced. Do not allow the column to dry. It is important to refill column voids at the top of the column with either glass beads (200 to 400 mesh) or residual packing material. A guard column or precolumn filter can be used. If a guard column is used, replace it and check backpressure before reversing ion-exchange column.

    Detection of Proteins
    Detection should be at 210 to 220 nm, where the peptide bond absorption occurs. The absorption is proportional to the number of peptide bonds in a protein. Detection at 280 nm may be useful for peptides or proteins containing chromophores (i.e., Tyr or Trp). Detection can be achieved using fixed-wavelength, variable-wavelength, or photodiode array detectors. A fixed-wavelength detector may be cheaper than the variable-wavelength detector, although in the long run the latter is a better investment. The photodiode array detector has the advantage of recording a complete spectrum at each time point, but is 10-fold less sensitive than other conventional detectors.




    MATERIALS AND SOLUTIONS

    Preparation of HPLC Buffer
    All buffer salts, organic solvents, chaotropic agents, detergents, and water must be of high purity, preferably HPLC grade. Buffer pH must be carefully adjusted to within 0.1 unit to ensure reproducibility of ion-exchange chromatography. pH adjustment should be done after all ingredients have been dissolved and after the solutions have been filtered. To minimize bacterial growth in ion-exchange buffers, 2 to 5% methanol should be added to each buffer, and when not in use, columns should be stored in a refrigerator. Laboratory water should be filtered and degassed. As a matter of practice, buffers should be prepared fresh daily. Any solution containing buffer salts should be filtered through a 0.45 um nylon filter into the flask. If volatile buffers or organic solvents are used, these should be added after filtration.


    Preparation of Protein Sample
    Proteins must be fully solubilized in a buffer of identical pH and lower ionic strength than the starting buffer of the gradient separation. If visual inspection indicates insolubility, then urea, organic solvent (e.g., methanol or propanol), or detergent (SDS or CHAPS) can be added to solubilize the protein. All samples should be centrifuged prior to injection in order to remove insoluble particulates.


    Ion-Exchange HPLC Protein Standards
    Ovalbumin --------------------------------------------- 5 mg
    alpha-Chymotrypsinogen ------------------------------- 5 mg
    Ribonuclease A ---------------------------------------- 5 mg
    Lysozyme ---------------------------------------------- 5 mg
    alpha-Lactalbumin -------------------------------------- 2.5 mg
    Distilled H2O ------------------------------------------- 1 ml
    Incubate at room temperature for 1 hour.

  • For anion exchange HPLC, dilute the Protein Standard Solution 10-fold with 0.02 M Tris-HCl (pH 7.5).
  • For cation exchange HPLC, dilute the Protein Standard Solution 10-fold with 0.02 M Sodium Phosphate (pH 6.0).


    0.2 M Tris-HCl (pH 7.5)


    Tris/ NaCl Buffer (1 liter)
    0.02 M Tris-HCl (pH 7.5) ------------------------------ 20 ml of 1 M Tris-HCl
    0.5 M NaCl -------------------------------------------- 100 ml of 5 M NaCl
    Distilled H2O ------------------------------------------- 1 ml


    0.2 M Sodium Phosphate (pH 6.0)


    Sodium Phosphate/ NaCl Buffer
    0.02 M Sodium Phosphate (pH 6.0) -------------------- 20 ml of 1 M Sodium Phosphate
    0.5 M NaCl -------------------------------------------- 100 ml of 5 M NaCl
    Distilled H2O ------------------------------------------- 1 ml




    PROCEDURES







    NOTES

  • An HPLC column can irreversibly adsorb protein until all its adsorption sites have been coated with protein. This can be a major source of protein loss during HPLC separations. For this reason guard columns are generally not recommended.

  • Since many organic and inorganic compounds absorb light in the far UV200-220 nm, it is necessary to use buffer chaotropic agents, organic solvents, detergents, and water that are HPLC grade and do not absorb strongly in this region.

  • The glassware used to prepare and store buffers should be detergent-free because trace amounts of detergent can lead to significant contamination.




    KIT INFORMATION




    REFERENCES

  • Chang SH, Csooding KM, Regnier FE (1976) High-performance liquid chromatography of proteins. J. Chromatogr. 125:103-114.

  • Nakanitira K, Kato Y (1985) Preparative high-performance ion-exchange chromatography. J. Chromtography. 333:29-40.

  • Peterson EA, Chiazzc EA (1962) Some experimental factors in the gradient chromatography of serum proteins. Arch. Biochem. Biophys. 99:139-144.

  • Regnier F (1984) High-Performance ion-exchange chromatography. Meth. Enzymol. 104:170-189.


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