Ion-Exchange Chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre-and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80 kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

Gel-filtration chromatography is a popular and versatile technique that permits the effective separation of proteins and other biological molecules in high yield. Here, the basis of the method is described and typical matrix types are contrasted. The selection of suitable operating conditions and applications of the method are also discussed.

Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

A group of enzymes exists that specifically recognises proline within proteins and peptides. Prolyl endopeptidase is one such enzyme, which cleaves on the carboxyl side of proline within peptide substrates. Its broad specificity towards bioactive peptides has led to its implication in various disease states including neurodegenerative and psychiatric disorders. This association has been based primarily on the abnormal levels of activity observed following the enzymes detection with the reportedly specific fluori‐metric substrate, 7‐(N‐benzyloxycarbonyl‐glycyl‐prolyl‐amido)‐4‐methylcoumarin (Z‐Gly‐Pro‐NH‐Mec). In this study, we report the discovery and preliminary characterisation of a Z‐Gly‐Pro‐NH‐Mec‐hydrolysing activity that is distinct from prolyl oligopeptidase (prolyl endopeptidase). Following the production of serum from bovine whole blood, Z‐Gly‐Pro‐NH‐Mec hydrolysis in serum was determined to be 7.2 U/mg protein. In the presence of 350 nM Z‐Pro‐prolinal, a specific inhibitor of prolyl endopeptidase, residual Z‐Gly‐Pro‐NH‐Mec hydrolysis of 2.6 U/mg protein was observed. This residual activity was resistant to inhibition by Z‐Pro‐prolinal at concentrations in excess of 200 times its reported Ki value for prolyl endopeptidase and could not be inhibited under conditions of prolonged incubation with the inhibitor. Following cation‐exchange chromatography, Z‐Gly‐Pro‐NH‐Mec‐hydrolysing activity was resolved into two distinct entities. The first of these activities was inhibited by Z‐Pro‐prolinal, demonstrated activity towards the thyroliberin analogue, 7‐(pyroglutamyl‐histidyl‐prolyl)‐4‐methylcoumarin (Glp‐His‐Pro‐NH‐Mec), and was catalytically enhanced under reduced assay conditions. This activity was subsequently designated prolyl endopeptidase. The second activity was totally resistant to Z‐Pro‐prolinal inhibition, demonstrated no activity towards Glp‐His‐Pro‐NH‐Mec, and was unaffected when assayed under reduced conditions. It was subsequently designated Z‐Pro‐prolinal‐insensitive Z‐Gly‐Pro‐NH‐Mec‐hydrolysing peptidase (ZIP).

Most proteins and large polypeptides have hydrophobic regions at their surface.These hydrophobic 'patches' are due to the presence of the side chains of hydrophobic or non-polar amino acids such as phenylalanine, tryptophan, alanine and methionine. These surface hydrophobic regions are interspersed between more hydrophilic or polar regions and the number, size and distribution of them is a specific characteristic of each protein.Hydrophobic Interaction Chromatography (HIC) is a commonly used technique that exploits these hydrophobic features of proteins as a basis for their separation even in complex biological mixtures (1) (2). In general the conditions under which hydrophobic interaction chromatography is used are relatively mild and 'protein friendly' resulting in good biological recoveries. Hydrophobic binding is relatively strong and is maintained even in the presence of chaotrophic agents, organic solvents and detergents. For these reasons this technique has a widespread use for the purification of proteins and large polypeptides.