Ion-exchange chromatography: basic principles and application to the partial purification of soluble mammalian prolyl oligopeptidase.
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).
Cummins
Philip M
PM
School of Biotechnology, Dublin City University, Dublin, Ireland.
Dowling
Oonagh
O
O'Connor
Brendan F
BF
D000327:Adsorption; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000166:cytology; D002021:Buffers; D002417:Cattle; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003600:Cytosol / Q000737:chemistry; D004983:Ethanolamines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility
D002021:Buffers; D004983:Ethanolamines; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; C007369:2-diethylaminoethanol; D000645:Ammonium Sulfate