Use of ion-exchange Sep-Pak cartridges in the batch fractionation of pituitary peptides

Abstract

Silica-based ion-exchange Sep-Pak cartridges, packed with either carboxymethyl (CM) cation-exchanger or a quaternary methyl ammonium (QMA) anion exchanger, are now available. The feasibility of using ion-exchange Sep-Pak cartridges for the fractionation of pituitary peptides was investigated. Extracts of bovine posterior pituitaries were fractionated at either pH 5 or pH 7 by pairs of cation and anion exchangers, connected in series. The capacity to bind peptides was well correlated with the theoretical charge calculated for a variety of peptides. At pH 5 the entire tissue extract could be fractionated into either basic or acidic pools. In contrast, at pH 7 only the more basic or acidic peptides were retained by the respective ion exchangers. The rest of the peptides passed through both ion exchangers and were recovered in the neutral pool. The ion-exchange fractionation principle was used to facilitate the purification of 35S-labelled intermediate pituitary glycopeptides, prepared by incubating mouse intermediate lobes in explant culture with 35S-labelled sulphate. 35S-labelled glycosylated forms of Lys1 gamma 3MSH, corticotropin-like intermediate lobe peptide, and the amino terminal or 16K fragment of pro-opiomelanocortin (i.e. 16K1-74) were fractionated into separate pools such that they could be purified to homogeneity in a single step by reversed-phase high-performance liquid chromatography (RP-HPLC). Purification by conventional means would require at least two RP-HPLC steps. Thus, radiolabelled peptides can be purified with the minimum of chromatographic manipulation, thereby ensuring maximal recoveries.