In gel permeation chromatography proteins do not bind significantly to the matrix. Sorting does not occur as a result of differential binding but of differential exclusion from pores in the beads. On the other hand many sorbents are available that bind proteins with different affinities. At equilibrium the behavior of a specific protein can be described by a partition coefficient a, the fraction of the protein that is adsorbed. a therefore can have values between 0 (no binding whatever) and 1.0 (complete adsorption without any desorption). Differences in partition coefficients can be utilized for fractionation by allowing proteins to choose between a stationary, adsorbed and a mobile, unadsorbed phase. In this case the completely unadsorbed protein will move with the buffer front; its mobility compared to the buffer front, R_f, will be unity. In contrast, the completely absorbed protein will not move at all, and R_f will be 0. In other words: a + R_f = 1.0. Binding of protein to the matrix is most simply achieved by mixing them. Such so-called batch adsorption is especially popular with hydroxyapatite, but can also be performed with ion exchange resins. One of the great advantages of adsorptive methods is the ability to use high volumes of sample, obviating concentration steps. This saves time and generally leads to higher overall yields in protein preparations. For example, one can load ion exchange resins in batch mode by stirring a large-volume sample with the resin. Subsequently, the resin is poured into a column for gradient elution, or batch eluted on a sintered glass funnel. Thus, one can move rapidly through large volume steps at the early stages of the preparation.

(i) Ion exchange chromatography: In this type of chromatography, the charged groups on the surface of a protein bind to an insoluble matrix with opposite charge. More precisely, the ionized protein displaces the counterions (e.g., chloride or sodium) of the matrix functional group, and will itself be displaced by an increasing concentration of ions in the elution buffer. Alternately, a pH gradient may be employed so that the net charge on the adsorbed protein decreases. Under specific starting conditions of buffer, pH, and ionic strength, the net charge on the protein of interest can be manipulated to interact with the matrix. Thus, the most important parameters to consider in an ion exchange separation are the choice of ion exchange matrix and the initial conditions. The ion exchange matrix: Ion exchangers consist of an insoluble containing charged groups. Anion exchangers contain basic groups that are positively charged below their pK's (e.g. amino functions) and cation exchangers contain acidic groups that are negatively charged above their pK's (e.g. carboxylate). For example, carboxymethyl is a weakly acidic cationic exchanger, while sulfopropyl is a strong cation exchanger, since the pK for the acidic proton is lower for sulfate than for acetate, i.e. the sulfopropyl function, i.e. the sulfopropyl function remains chraged over a greater part of the pH range than the carboxymethyl. A cationic protein will bind to both types of cation exchangers, but it will usually require higher concentration of counter ions to desorb the protein from the "stronger" ion exchanger, sulfopropyl. Similarly, DEAE is a weakly basic anion exchanger, while the quaternary ammonium ions are strong anion exchangers. A special case of ion exchange chromatography is metal chelation chromatography. Here resins contain carboxylate functions in close proximity which are capable of making coordination complexes with metal ions such as Ni²⁺. The metal cations are then capable of interacting strongly with strings of imidazols, i.e. histidine residues in proteins. The strong coordination bond can be broken by an excess of free imidazol. This type of chromatography is especially useful for the isolation of proteins under denaturing conditions, provided a string of histidines is present. Such a His tag is easily engineered into fusion proteins.

(ii) Hydrophobic interaction chromatography: In hydrophobic interaction chromatography (HIC) advantage is taken of hydrophobic patches on the protein surface that interact with non-polar materials under non-denaturing conditions. As in ammonium sulfate fractionation, high ionic strength promotes interactions between surface hydrophobic patches on the proteins. For loading, the ionic strength is raised to just below the point of precipitation in the presence of an immobilized hydrophobic matrix such as phenyl or octyl agarose (note that plain agarose or Sephadex will do, as hydrophobic interactions with the polysaccharide matrix in the absence of alkyl or aryl substituents are possible).. Ammonium sulfate concentrations of 1 M (ca 25% saturation) are usually sufficient for proteins to bind to the matrix rather than precipitate. A descending salt gradient then causes desorption of proteins in the order of hydrophobic affinity between matrix and protein surface. At very low ionic strength, there may still be proteins retained by the column, and these must be removed by the addition of nonpolar or chaotropic components to the matrix. Solvent modifiers added to cause desorption of strongly bound proteins can lead to denaturation of proteins. Under those conditions, HIC becomes similar to reverse phase systems using organic solvents (Notice the similarity to protein precipitation methods, this time solvent precipitation, and to reverse phase HPLC, which is widely used in the fracionation of peptides).

There are many ways to covalently link suitable ligands to resin beads. One of the most popular is the activation of agarose resins with cyanogen bromide; the activated resin contains cyanate esters which react with amino functions to give isourea derivatives. The chemistry of this classical method is described on the handout (Note that according to Porath (Meth. Enz. 34, 13-30, 1974) imido carbonates are the reactive intermediates, whereas Scopes says that it is the cyanate ester which couples to amino groups to generate isourea derivatives. At any rate the leakage problem has been attributed to the instability of both isourea and imidocarbonate derivatives - Parikh et al., Meth. Enz. 34, 77-108, 1974).

Examples of other immobilization chemistries:

aldehyde functions for reaction with NH₂

(Schiff base formation)

epichlorohydrin, bisoxirane for reaction with -NH₂, -OH, -SH

(formation of stable secondary amines,

ethers and thioethers; epichlorohydrin

is used to reinforce dextran-based resins!)

tosyl-, tresyl- activated resins for reaction with NH₂

(formation of stable secondary amines)

N-hydroxysuccinimide esters for reaction with NH₂

(formation of stable amides)

carbodiimide for the activation of carboxyl functions

diazo coupling Used in early phases, e.g. for the preparation of immunosorbents

It is worth mentioning that many of the resulting links (e.g. isourea derivatives arising from CNBr activation or Schiff bases arising from the reaction of aldehydes with amino groups) are not stable, especially at high pH, and therefore have a tendency to leak or ‘bleed’. This is particularly troublesome if rare proteins that have a high affinity for the ligand are to be fractionated, because even at fairly slow bleeding rates sufficient free ligand will be available within the resin to bind to the protein and prevent it from being adsorbed to the resin. Similarly, effects of bead-coupled proteins on intact cells can never be claimed to be caused by proteins acting extracellularly, since a small fraction may have been released during the experiment. Prolonged and rigorous washes of the newly-synthesized resins are a must!

Ligand Application

L-arginine serine proteases

L-lysine plasminogen, plasminogen activator, rRNA

p-aminobenzamidine serine proteases (benzamidine is used as a blocker of trypsin-like proteases

Cibacron Blue Enzymes requiring NAD & NADP, albumin, coagulation factors, interferon, a 2 macro- globulin (Note that other dyes also have been used as group-specific ligands; an example is Procion Red for the purification of NADP-dependent enzymes and carboxypeptidase G)

calmodulin kinases, phosphatases, other calmodulin- dependent enzymes

gelatin fibronectin

glutathione glutathione-S-transferase (GST), GST fusion proteins

heparin growth factors, steroid receptors, DNA binding proteins, restriction enzymes, lipases, lipoproteins

protein A mammalian IgG (human, rabbit) (Note that some mouse IgG classes bind better to protein G, another Staphylococcus protein)

Lectins: concanavalin, lentil glycoproteins containing D-glucose

lectin and D-mannose

Ricinus communis lectin D-Gal

wheat germ agglutinin D-GlucNAc

Glycine max, Arachis

hypogaea lectins

Helix pomatia, Dolichos D -GalNAc

bifloris, Glycine max,

Arachis hypogaea lectins

Lotus tetragonolobus L-Fucose

Ulex europeus

(Immunosorbents antigens, antibodies) ~ were the first affinity resins to be prepared