FILTRACIÓN APLICADA AL TRATAMIENTO DE EFLUENTES

The crystallisation of biological macromolecules is a multi-parametric process involving the three steps of nucléation, growth and cessation of growth. The main difference from small molecule crystal growth arises from the conformational flexibility and chemical versatility of macromolecules and their consequent greater sensitivity to extemal conditions. For a rational design of growth conditions, physical and biological parameters need to be controlled.

2.4.1 Purity

Purity is not an absolute requirement for crystallisation as crystals can be obtained from mixtures. Poor purity is however unlikely to result in high quality monocrystals of the size required for the purpose of X-ray crystallography. Typically crystals should be of the order of 0.2 mm in size, but the use of synchrotron radiation sources and cryogenics means that crystals of much smaller dimensions may yield data suitable for structural analysis. For stmctural analysis, the macromolecular sample has to be of high purity, not only in terms of lack of contaminants but also conformationally pure. Denatured macromolecules or macromolecules with stmctural microheterogeneities adversely affect crystal growth more than unrelated molecules do.

2.4.2 Solubilities and supersaturation

a supersaturated, thermodynamically unstable state, which may develop into a crystalline or amorphous phase when it returns to equilibrium. Supersaturation can be achieved by slow evaporation of the solvent or by varying specific parameters. Intrinsic physical and chemical parameters that affect the protein solubility are : the concentration of protein and précipitants; temperature; pH; rate of crystal growth; ionic strength and purity of chemicals; volume and geometry of samples; wall and interface effects; density and viscosity; pressure; electric and magnetic fields; vibrations and sound; and the sequence of events that arrive at a specific set of parameters. Biochemical parameters may include the sensitivity of the protein conformations to physical parameters, the binding of ligands, specific additives such as reducing agents, polyanionic detergents and polyamines. These are also related to the properties of the protein. The source and age of the protein preparation are also parameters that may affect solubility. The solubility of a macromolecule can be represented in a phase diagram as a function of one parameter, all other parameters being constant (Figure 2.3).

The solubility curve divides the under-saturated and supersaturated zones. Under the solubility curve the protein will never crystallise. Above the solubility curve the concentration of the protein is higher than the concentration at equilibrium for the given parameter. This corresponds to the supersaturation zone. The level of saturation is defined as the ratio of protein concentration over the solubility value. Depending on the kinetics to reach equiUbrium and the level of supersaturation, this region may be subdivided into three zones. The precipitation zone is where excess protein immediately separates from the solution in an amorphous state. The nucléation zone is where excess protein separates into a crystalline form. Near the precipitation zone, crystallisation may occur as a shower of tiny microcrystals which can be confounded with amorphous precipitate. In a metastable zone a supersaturated solution may not nucleate for a long period of time, unless the solution is mechanically shocked or a seed crystal is introduced. This zone corresponds to the growth of crystals without the nucléation of new crystals.

c 0

1

o

a

supersaftiration

precipitation zone

nucléation zone

metastable zone

solubility curve

undersaturation

Salt concentration

Figure 2.3 Phase diagram representing the solubility of a protein as a function of a single parameter, in this case the salt concentration.

2.4.3 Nucléation and cessation of growth

Proteins require defined pH and ionic strength for stability and function and so ciystaUisation often requires complex aqueous solutions. Crystallisation starts with a nucléation phase followed by a growth phase and nucléation requires a greater supersaturation than the growth phase. Cessation of growth occurs when the protein has been depleted from the medium, or because of growth defects, poisoning of the faces or ageing of the protein. The crystal quality may be correlated with the packing of the molecules into the crystal lattice and the external crystal morphology. The forces that are involved in the packing of macromolecules are weak compared to those maintaining the cohesion of small-molecule crystals. These forces involve salt bridges, hydrogen bonds, van der Waals, dipole-dipole and stacking interactions. The weak cohesion of macromolecule crystals results fi*om the fact that only small parts of the macromolecular surfaces participate in intermolecular contacts, the remainder being in contact with the solvent. This explains the commonly observed polymorphism of biological macromolecular crystals.

2.4.4 Typical trial arrays

Screening the physical and chemical parameters required to achieve the specific conditions in which a particular macromolecule will crystallise can be a daunting task. Figure 2.4 summarises the systematic approach that was employed in the proj ects described in this thesis to grow protein crystals.

Matrices fi>cused on solubDity minima observed in inirial conditions

Concentrate protein S to 10 £)Id if possible.

Proteo^ücaDy modified protein

Empby heterogeneous or epitaxial nucleants, or nucleant c a ta l^ Purify protein fistber

Repeat with proteins fiom another source Metalions

eg Mg2+, Ni2+

Detergents