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4.1 Introduction.

Adsorption chromatography depends upon interactions o f different types between solute molecules and ligands, immobilised on a chromatographic matrix. This chapter described the chromatographic media and method scouting experimentation, conducted to assess the most suitable mechanism of adsorption for the capture o f the target enzyme, ADH, from crude feedstocks.

Three type of packed bed Sepharose based matrices were examined; ion-exchange, hydrophobic interaction and immobilised metal ion affinity chromatography. In all three cases, initial experimentation was first conducted using solutions o f the pure enzyme. Following the optimised capture o f the pure enzyme, the complexity o f the solution from which the ADH was capture was increased by attempting to isolate the enzyme from a solution of clarified yeast supernatant. The performance of all three types o f adsorption mechanism were compared and the most suitable form was

selected for further experimentation.

Before the results from the method scouting experimentation are presented, there follows a short introduction to each o f the three type o f adsorption investigated. The salient theoretical considerations and basic principles for each type are also briefly presented in the following sections with greater coverage and depth allocated to those areas which are accompanied by a large number o f experimental results.

4.1.1 Ion-exchange chromatography.

Historically, ion-exchange chromatography was introduced with the cellulose ion exchangers in the mid 1950's by Sober and Peterson (Sober and Peterson, 1954; Peterson and Sober, 1956; Sober et a l, 1956). They synthesised the (DEAE) Diethylaminoethyl and (CM) Carboxymethyl derivatives o f cellulose still in use today. Ion-exchange chromatography is by far the most widely used form o f chromatography, included in about 75 % o f purification protocols (Bonnerjea et a l,

reason for its popularity is its versatility, high resolving power, high capacity and straight forward basic principle

4.1.1.1 Theory.

Proteins bind to ion exchangers by electrostatic forces between the proteins' surface charges and the dense clusters o f charged groups on the exchangers. The charges on the ion exchanger are balanced by counter ions such as metal ions, chloride ions and buffer ions. A protein must displace the counter ions to become attached. Generally the net charge on the proteins will be the same sign as the counter ions displaced- hence ion exchange. The protein molecules in solution are also neutralised by counter ions, the overall effect in a given region o f the adsorbent must be electrically neutral. The interaction between the protein and the ion exchanger depends upon several factors;

• net charge and surface charge distribution of the protein

• the ionic strength and nature of the particular ions in the solvent • pH

• other additives to the solvent, i.e. organic solvents

The energy gained by the formation o f an ionic bond between a protein and a charge on the stationary phase is expressed by the coulombic law:

4.1

K . Y A E

where AE is the change in energy as two charges A and B with Z a and Z g number of unit charges are brought within a distance of r ^ . k is the dielectric constant o f the medium. If the two charges are o f opposite sign, there is a decrease in energy and if o f the same sign, an increase in energy. It is clear that the more highly charged a protein is, the more strongly it will bind to a given oppositely charged ion exchanger. Similarly more highly charged ion exchangers; those with a higher degree of substitution o f charged groups, bind proteins more effectively than weakly charged ones. Conditions which alter the effective charge on either the protein or the ion exchanger, such as pH or ionic strength, will affect their interaction and are used to influence the ion exchange process.

4.1.1.2 Influence of pH on adsorption.

pH is one of the most important parameters which controls protein binding as it determines the effective charge on both the protein and the ion exchanger. If reproducible results are to be obtained, control o f the pH by the addition of buffer salts is essential. Although proteins have charges of both signs over a wide range o f pH, as a rule binding to an ion exchanger only occurs when there is a net charge o f opposite sign to that possessed by the ion exchanger. At pH values far away from the proteins' pi, proteins bind strongly and in practice do not desorb at all at low ionic strengths. At pH’s near to the pi, the protein has no net charge and binding is correspondingly weaker. There are situations where binding still occurs even when the protein and the ion exchanger have the same net charge (Kopaciewiecz 1983). This is due to the uneven distribution o f the charged groups across the surface o f the protein.

4.1.1.3 Influence of ion concentration.

The binding o f proteins to charged groups on the stationary phase competes with the binding o f other ions within the solvent. At low enough concentrations binding o f proteins occurs through multiple charge interactions between several groups on the protein and a corresponding number, the Z-number o f charges on the ion exchanger. At higher concentrations o f competing ions, i.e. a higher ionic strength, the proteins will start to be displaced from the ion exchanger in order o f their binding strength. There is no general rule as to what salt concentration is required to displace a protein with a given charge. However most proteins are eluted at salt concentration o f between 0.01 and 0.5M. The type of ion is also an important factor in the process and ions which interact specifically with charged groups on the protein will be unusually effective in elution.

4.1.1.4 Matrices and functional groups for ion-exchangers.

Ion-exchange matrices consist o f a matrix substituted with either basic or acidic groups. The basic ion exchangers containing positive groups are called anion exchangers while the acidic ones containing negative groups are called cation exchangers. The matrices are either;

• resins-hydrophobic polystyrene bases or partly hydrophobic polymethacrylate based polymers

• hydrophilic synthetic and naturally occurring polymers such as cellulose, dextran or agarose etc.

• various synthetic hydrophilic polymers which make hard beads for high pressure applications.

• silica gels

The beaded forms of dextran and agarose gels originally prepared for gel filtration have been derivatized to produce ion exchangers for protein separation work. The Sephadex™ based ion exchangers (Pharmacia Biotech AB, Sweden) are derived from dextran. The Sepharose™ ion exchangers are derived from 6 % crosshnked agarose (Sepharose™ CL-6B). These gels are more porous than the dextran gels and new developments of these media, Q- and S-Sepharose™ are produced by a different crosslinking procedure resulting in beads that are the same size but more rigid and suitable for large scale work and other applications for which a high flowrate is required. Agarose based ion exchangers are particularly suitable for chromatography of larger proteins as they are quite macroporous.

4.1.2 Hydrophobic interaction chromatography.

Hydrophobic molecules in aqueous solvents will self-associate due to hydrophobic interaction. In biological systems, hydrophobic interaction is of great importance as the folding o f globular proteins, association o f protein subunits and many other biological processes rely on hydrophobic interaction for their correct function. Hydrophobic interaction chromatography utilises the fact that the surfaces o f globular proteins have extensive hydrophobic patches in addition to the expected hydrophilic groups. These hydrophobic regions bind to the hydrophobic ligands, alkyl or aryl side chains on the gel matrix under conditions favouring interaction for example aqueous solutions with high salt concentrations.

The first attempt to synthesis hydrophobic adsorbents was made by Yon (1972) followed by Er-el et a l (1972), Hofstee (1973) and Shaltield and Er-el (1973). Characteristically these early adsorbents exhibited a mixed ionic-hydrophobic character (Wilchek and Miron, 1976). Despite this Halperin et a l (1981) claimed that protein binding to such adsorbents was predominately o f a hydrophobic nature. Later, Porath et a l (1973) and Hjerten et a l (1974) succeeded in synthesising charge-free

hydrophobic adsorbents and demonstrated that the binding o f proteins was enhanced by high concentration of neutral salts, confirming the earlier observation of Tiselius (1948), and that elution of the proteins fi*om the matrices could be achieved by washing the column with salt free buffer or by decreasing the polarity o f the eluent (Hofstee, 1973; Porath e /a /., 1973).

The first commercially produced hydrophobic adsorbents were the Octyl and Phenyl Sepharose C1-4B matrices (Janson and Laas, 1978) of the charge free type. The commercial availability o f new matrices, opened up the application o f hydrophobic techniques, to include a wide variety o f biomolecules such as serum proteins (Hrkal, Rejnkova, 1982), receptors (Kuehn et a l, 1980) and cells (Hjerten, 1981).

4.1.2.1 Theory of hydrophobic interaction chromatography.

The discussion that follows in this section will be limited to the non-charged type of HIC adsorbents as used in the later method scouting experiments.

A large number of theories have been proposed for the explanation o f hydrophobic interaction chromatography. However most o f the theories are essentially based upon the interaction o f hydrophobic solutes and water (Tanford, 1973; Creighton, 1984). None of the theories have enjoyed universal acceptance but common to them all is the central role o f structure-forming salts and the influence they exert on the solute, solvent and adsorbent within the chromatography system. Porath (1986) proposed “salt-promoted adsorption” as a general concept for HIC and other types o f solute- adsorbent interactions occurring in the presence o f high concentration o f neutral salts. Hofstee (1973), and later Shaltield and Er-el (1973), proposed the theory of “hydrophobic chromatography” with the implicit assumption that the mode o f interaction between proteins and immobilised hydrophobic ligands was similar to the self association of small aliphatic organic molecules in water. Porath et a l (1973) suggested a salting out effect in hydrophobic adsorption, extending the earlier observation o f Tiselius (1948). They suggested that “ ...the driving force is the entropy gain arising from structure changes in the water surrounding the interacting hydrophobic groups”. This concept was further extended and formalised by Hjerten (1977) who based this theory on the well known thermodynamic relationship: