Section 4.1.4.2. Purity and homogeneity

The concept of purity assumes a particular importance in crystallogenesis (Giegé et al., 1986; Rosenberger et al., 1996), even though some macromolecules may crystallize readily from impure solutions (Judge et al., 1998). In general, macromolecular samples should be cleared of undesired macromolecules and small molecules and, in addition, should be pure in terms of sequence integrity and conformation. Contaminants may compete for sites on growing crystals and generate growth disorders (Vekilov & Rosenberger, 1996), and it has been shown that only p.p.m. amounts of foreign molecules can induce formation of non-specific aggregates, alter macromolecular solubility, or interfere with nucleation and crystal growth (McPherson et al., 1996; Skouri et al., 1995). These effects are reported to be reduced in gel media (Hirschler et al., 1995; Provost & Robert, 1995).

Microheterogeneities in purified samples can be revealed by analytical methods, such as SDS–PAGE, isoelectric focusing, NMR and mass spectroscopy. Although their causes are multiple, the most common ones are uncontrolled fragmentation and post-synthetic modifications. Proteolysis represents a major difficulty that must be overcome during protein isolation. Likewise, nucleases are a common cause of heterogeneity in nucleic acids, especially in RNAs that are also sensitive to hydrolytic cleavage at alkaline pH and metal-induced fragmentation. Fragmentation can be inhibited by addition of protease or nuclease inhibitors during purification (Lorber & Giegé, 1999). Conformational heterogeneity may originate from ligand binding, intrinsic flexibility of the macromolecule backbones, oxidation of cysteine residues, or partial denaturation. Structural homogeneity may be improved by truncation of the flexible parts of the macromolecule under study (Price & Nagai, 1995; Berne et al., 1999).