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Salt-promoted adsorption chromatography
Journal of Chromatography, 510 (1990) 47-48 Elsevier Science Publishers B.V., Amsterdam CHROM.
22 367
Extended Abstract
Salt-promoted adsorption chromatography JERKER
PORATH
Separation Centre. Box 577. S-7SI 23 Uppsala (Sweden)
Molecules surrounded by organized water tend to self-associate and form molecular complexes with other kinds of molecules which also have hydration shells of water that are more organized than in the aqueous bulk phase. Formation constants for such complexes are to a greater or lesser extent governed by the entropic gain accompanying the reorganization of water following complexation. If one of the interacting species, the ligand, is surface bound to the support and the other, the ligate, is present in the surrounding solution, the latter will become adsorbed on the solid support. Three kinds of adsorption or affinity chromatography based on this principle and with interaction strength and capacity thereby promoted by water-structuring (anti-chaotropic) salts were briefly described: (1) HIC (hydrophobic interaction chromatography; (2) EDAC (electron donor-acceptor chromatography including thiophilic affinity chromatography; and (3) TAC and IMAC (immobilized metal ion affinity-based chromatography). The discussion is confined to peptide and protein fractionation. In a narrow sense, hydrophobic interaction of protein molecules refers to the mutual attraction of alkyl side-chains in water. Intermolecular interactions between alkyl ligands and alkyl ligates are exploited in hydrophobic interaction chromatography. If an aliphatic ligand is unsaturated, as is the case for propargyl-liganded agarose, another kind of interaction is superimposed on hydrophobicity. Likewise, phenyl or naphthyl ligands attract unsaturated ligates more strongly than can be accounted for by hydrophobicity alone. The additional contribution to the interaction is due to the rc-electron system of the ligand. The electron donor or acceptor strength of a ligand can be strongly enhanced by introduction of electron-releasing or -attracting substituents so that the hydrophobic character will play only a secondary role. This is the case if several groups are located adjacent to double bonds or are suitably located in heterocyclic rings. Nitro groups introduced in a benzene nucleus deplete the latter of electrons, making the support an electron acceptor adsorbent. On the other hand, a dimethylamino group enhances the electronegativity of the nucleus. Groups in proteins most likely aromatic side-chains, are the counterparts in the formation of adsorption complexes with these kinds of ligands. EDA adsorption may be further strengthened in the presence of high concentrations of alkaline and ammonium phosphates, sulphates, chlorides and other water-structuring salts.
48
J. PORATH
A non-ionic ligand may interact with a protein without being in a narrow sense hydrophobic, and still the interaction may be strongly salt-promoted. The first ligand of such a kind that we discovered has the simple structure -OCH2CH2S02CH2CH2SCH2CH20H. Owing to its sulphur content, we call the ligand thiophilic, and the corresponding agarose gel was named “T-gel”, where T represents either thiophilic or thioether. Used with buffer solutions, 0.3-0.65 Min K2S04, the T-gel turned out to be an excellent adsorbent for immunoglobulin isolation. The underlying phenomenon seems to be more general than was at first thought. The ligand may not necessarily contain thioether sulphur. The protein counter-ligands are likely to be the surfaceexposed aromatic side-chains, particularly indole groups, The nature of the ligand and solid support and the composition of the solvent medium modulate the strength and selectivity also in the metal-protein attraction and, consequently the specificity and capacity of an IMA adsorbent. There are a wide variety of methods to promote the desorption of proteins in IMAC, such as including in the eluent additives ethylene glycol, surfactants, affinity-competing solutes etc., and changing the pH and temperature. Metal ions such as Co2+ Ni2+, Cu2+ and Zn2+ interact chiefly with imidazole, but also with surface-exposed thiol and in certain circumstances to a lesser extent with indole and terminal amino groups. Very efficient high-performance liquid chromatography of peptides and proteins has been achieved using pH and imidazole gradients for elution. The extent of adsorption and desorption depends on the concentration and the kind of water-structuring salts used, but the effects are often the opposite for “soft” and “hard” metal ions. Preliminary studies on IMAC for the isolation of calcium- and magnesiumbinding proteins and phosphoproteins indicate that “hard” metal ions, immobilized or included in the eluent, may offer a great advantage over conventional purification procedures. High selectivity may be achieved in spite of the fact that the interactions, as in ion-exchange chromatography, appears to be only ionic in nature. Presumably the protein counter ions are carboxylic and phosphate ester groups. It is worth pointing out that the separation of proteins in solutions of water-structuring salts can be made sequentially, without intermittent desalting, using in optional order the affinity principle hydrophobicity, thiophilicity and metal ion specificity. By use of gradients and selective eluting agents, a refinement in the resolution of complex mixtures may be obtained in each chromatographic step. Other advantages are that bacterial growth is prevented by the virtually complete elimination of essential metal ions and the high salt concentration, which also tends to stabilize the Adsorption and desorption may be effected at tertiary structure of proteins. physiological pH and at ambient temperature.
REVIEWS 1 2 3 4 5 6
J. Porath, Biotechnol. Prog., 3 (1981) 14. J. Porath and M. Belew. Trendr Biotechnol.. 5 (1987) 225. J. Porath, Trends Anal. Chem., 7 (1988) 254. E. Sulkowski, Bioassay& 10 (1989) 170. M. A. Vijayalakshmi,Trends Biotechnol., 7 (1989) 71. J. Porath, in T. W. Hutchens (Editor), Protein Recognition of Immobilized Ligands, UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 80, Alan R. Liss, New York, 1989, pp. 101-122.
22 367
Extended Abstract
Salt-promoted adsorption chromatography JERKER
PORATH
Separation Centre. Box 577. S-7SI 23 Uppsala (Sweden)
Molecules surrounded by organized water tend to self-associate and form molecular complexes with other kinds of molecules which also have hydration shells of water that are more organized than in the aqueous bulk phase. Formation constants for such complexes are to a greater or lesser extent governed by the entropic gain accompanying the reorganization of water following complexation. If one of the interacting species, the ligand, is surface bound to the support and the other, the ligate, is present in the surrounding solution, the latter will become adsorbed on the solid support. Three kinds of adsorption or affinity chromatography based on this principle and with interaction strength and capacity thereby promoted by water-structuring (anti-chaotropic) salts were briefly described: (1) HIC (hydrophobic interaction chromatography; (2) EDAC (electron donor-acceptor chromatography including thiophilic affinity chromatography; and (3) TAC and IMAC (immobilized metal ion affinity-based chromatography). The discussion is confined to peptide and protein fractionation. In a narrow sense, hydrophobic interaction of protein molecules refers to the mutual attraction of alkyl side-chains in water. Intermolecular interactions between alkyl ligands and alkyl ligates are exploited in hydrophobic interaction chromatography. If an aliphatic ligand is unsaturated, as is the case for propargyl-liganded agarose, another kind of interaction is superimposed on hydrophobicity. Likewise, phenyl or naphthyl ligands attract unsaturated ligates more strongly than can be accounted for by hydrophobicity alone. The additional contribution to the interaction is due to the rc-electron system of the ligand. The electron donor or acceptor strength of a ligand can be strongly enhanced by introduction of electron-releasing or -attracting substituents so that the hydrophobic character will play only a secondary role. This is the case if several groups are located adjacent to double bonds or are suitably located in heterocyclic rings. Nitro groups introduced in a benzene nucleus deplete the latter of electrons, making the support an electron acceptor adsorbent. On the other hand, a dimethylamino group enhances the electronegativity of the nucleus. Groups in proteins most likely aromatic side-chains, are the counterparts in the formation of adsorption complexes with these kinds of ligands. EDA adsorption may be further strengthened in the presence of high concentrations of alkaline and ammonium phosphates, sulphates, chlorides and other water-structuring salts.
48
J. PORATH
A non-ionic ligand may interact with a protein without being in a narrow sense hydrophobic, and still the interaction may be strongly salt-promoted. The first ligand of such a kind that we discovered has the simple structure -OCH2CH2S02CH2CH2SCH2CH20H. Owing to its sulphur content, we call the ligand thiophilic, and the corresponding agarose gel was named “T-gel”, where T represents either thiophilic or thioether. Used with buffer solutions, 0.3-0.65 Min K2S04, the T-gel turned out to be an excellent adsorbent for immunoglobulin isolation. The underlying phenomenon seems to be more general than was at first thought. The ligand may not necessarily contain thioether sulphur. The protein counter-ligands are likely to be the surfaceexposed aromatic side-chains, particularly indole groups, The nature of the ligand and solid support and the composition of the solvent medium modulate the strength and selectivity also in the metal-protein attraction and, consequently the specificity and capacity of an IMA adsorbent. There are a wide variety of methods to promote the desorption of proteins in IMAC, such as including in the eluent additives ethylene glycol, surfactants, affinity-competing solutes etc., and changing the pH and temperature. Metal ions such as Co2+ Ni2+, Cu2+ and Zn2+ interact chiefly with imidazole, but also with surface-exposed thiol and in certain circumstances to a lesser extent with indole and terminal amino groups. Very efficient high-performance liquid chromatography of peptides and proteins has been achieved using pH and imidazole gradients for elution. The extent of adsorption and desorption depends on the concentration and the kind of water-structuring salts used, but the effects are often the opposite for “soft” and “hard” metal ions. Preliminary studies on IMAC for the isolation of calcium- and magnesiumbinding proteins and phosphoproteins indicate that “hard” metal ions, immobilized or included in the eluent, may offer a great advantage over conventional purification procedures. High selectivity may be achieved in spite of the fact that the interactions, as in ion-exchange chromatography, appears to be only ionic in nature. Presumably the protein counter ions are carboxylic and phosphate ester groups. It is worth pointing out that the separation of proteins in solutions of water-structuring salts can be made sequentially, without intermittent desalting, using in optional order the affinity principle hydrophobicity, thiophilicity and metal ion specificity. By use of gradients and selective eluting agents, a refinement in the resolution of complex mixtures may be obtained in each chromatographic step. Other advantages are that bacterial growth is prevented by the virtually complete elimination of essential metal ions and the high salt concentration, which also tends to stabilize the Adsorption and desorption may be effected at tertiary structure of proteins. physiological pH and at ambient temperature.
REVIEWS 1 2 3 4 5 6
J. Porath, Biotechnol. Prog., 3 (1981) 14. J. Porath and M. Belew. Trendr Biotechnol.. 5 (1987) 225. J. Porath, Trends Anal. Chem., 7 (1988) 254. E. Sulkowski, Bioassay& 10 (1989) 170. M. A. Vijayalakshmi,Trends Biotechnol., 7 (1989) 71. J. Porath, in T. W. Hutchens (Editor), Protein Recognition of Immobilized Ligands, UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 80, Alan R. Liss, New York, 1989, pp. 101-122.