Ionic strength

3.1.3.1 Salting-in

At low ionic strength, a phenomenon known as “ salting-in” occurs in which the solubility o f the protein decreases at very low ionic strength when the ions essential for satisfying the electrostatic requirements o f the protein molecules decreases. As these ions are removed, the protein molecules seek to balance their electrostatic requirements through interactions amongst themselves. Thus, they aggregate and separate from the solution. The salting-in effect, can be used as a crystallisation tool through dialysis. One can dialyse a protein soluble at moderate ionic strength against distilled water. Many proteins like catalase, concanavalin B and immunoglobulins have been crystallised by this means (McPherson, 1990).

3.1.3.2 Salting-out

Salting out is a phenomenon in which salt ions and macromolecules compete for the solvent molecules, that is, water. Both ions and proteins require hydration layers to maintain solubility (McPherson, 1990). The increasing salt concentration favours interaction between hydrophobic and other residues which exclude water. Hence, they tend to associate with themselves and aggregate. Thus dehydration or the perturbation o f solvent layers around the protein molecules, induces insolubility. The ionic strength, p, takes into account the valency, Zi, o f all ions present as well as the salt concentration (M i) in the solution as indicated in Equation [1] (Ries-Kautt and Ducruix, 1992).

H = l / 2 S ( M i Z i ^ ) [1]

Classical salts such as ammonium sulphate as well as phosphates and citrates have been used to increase the ionic strength (McPherson, 1990).

Theoretical considerations

3.1.3.3 Organic solvents and Polymers

The organic solvent can compete for water molecules and reduce their dielectric screening capacity. Reduction o f the bulk dielectric increases the effective strength o f the electrostatic forces allowing protein molecules to attract each other. Polymers such as poly (ethylene glycol) serve to dehydrate proteins in solution by altering the dielectric properties similar to organic solvents. They perturb the natural structure o f the solvent and create a more complex network having both water and itself as structural elements. A consequence o f this solvent restructuring is that macromolecules, particularly proteins, tend to be excluded and phase separation is promoted (McPherson, 1990).

3.1.3.4 Protein concentration

Proteins are kept in solution by a primary hydration sphere. This sphere o f water becomes limiting when the protein concentration is increased beyond some point. The protein starts aggregating and at the solubility limit, the protein will crystallise or precipitate. The protein concentration is an important parameter in protein crystallisation in that it is difficult to define the optimal protein concentration. For crystallisation trials, protein concentration o f 10 to 20 mg.mL'^ is sufficient. Proteins in the Biological Macromolecule Crystallisation Database (BMCD)

(Feigelson, 1988) have been crystallised from solutions containing less than 1.0 up to several hundred m g.m L '\

3.1.3.5 Purity

Crystal growth rates can be considered in terms o f the transport o f molecules to the growing nucleus and attachment o f them to the growing surfaces. The capture o f molecules by a growing crystal surface requires the molecules to be incorporated in the correct orientation and to be in a proper chemical state to form interactions with its neighbouring molecules (McPherson, 1990).

Crystal growth requires the population o f molecules to be as chemically homogeneous as possible. This means that contaminating proteins should be eliminated. Because crystals have perfect symmetry and periodic translational

Theoretical considerations

relationships between molecules in the lattice, non-uniform protein units cannot enter the crystal. Thus, contaminating impurities may serve as inhibitors o f growth.

If they enter the lattice, they may introduce imperfections which produce defects, dislocations and probably termination o f crystal growth. It has been recorded that the presence o f foreign substrates may facilitate nucléation (Gilliland,

1988). Crystals o f lipase from Candida rugosa have been crystallised from an impure solution by adjusting the pH to the pi o f the protein (U.S Patent, 1992).

3.1.3.6 Description of a phase diagram

As the solubility o f a biological macromolecule depends on the various parameters described, a two-dimensional solubility diagram is a representation o f its solubility (mg.mL'^ or mM macromolecule in solution ) as a function o f one

parameter, e.g. salt concentration, with all other parameters constant. The diagram comprises the following zones (Figure 4) (Ries-Kautt and Ducruix, 1992):

Protein concentration

SUPERSAT

TION

Undersaturated

Figure 4: Schematic description o f a two-dimensional solubility diagram, demonstrated by the change o f protein concentration with crystallising agent concentration (Gilliland, 1988).

Theoretical considerations

protein. This corresponds to the situation at the end o f the process o f crystal growth from a supersaturated solution.

(b) Under the solubility curve (S), the solution is undersaturated and the protein will never crystallise.

(c) Above the solubility curve (S), the macromolecule concentration is higher than the concentration at equilibrium for a given salt concentration. This corresponds to the supersaturation zone and is subdivided into three regions. (I) The precipitation zone is where excess protein immediately separates from the solution in an

amorphous state, (ii) The nucléation zone is where excess macromolecules separate under a crystalline form. Near the precipitation zone, crystallisation occurs as a shower o f microcrystals which can be confused with precipitate, (iii) In a metastable zone, a supersaturated solution may not nucleate unless the solution is mechanically shocked or a seed crystal introduced. This region corresponds ideally to the growth o f crystals with nucléation o f new crystals.

3.2

THE PHYSICAL CHEMISTRY OF PROTEIN CRYSTALLISATION