The combined use of size-exclusion and reversed-phase high-performance liquid chromatography for characterization of β-endorphin processing pathways

A method is described for the separation and analysis of multiple molecular forms of immunoreactive β-endorphin and its α-N-acetylated congenera by a combination of reversed-phase and size-exclusion high-performance liquid chromatography coupled with two specific radioimmunoassays. Both chromatographic procedures are fast ( < 50 min per analysis) providing good resolution and high recovery ( > 90% ). The solvents used in both systems are ultraviolet transparent ( < 214 nm), non-corrosive, low salt ( < 0.05 M) and after evaporation fully compatible with subsequent radioimmunoassay. We have evaluated these techniques using both synthetic and purified peptide standards and have applied these procedures to characterize immunoreactive β-endorphin and α-N-acetylendorphin in rat and sheep pituitary extracts, and the low levels found in sheep hypothalamus and rat ovary. These chromatographic procedures are not only applicable to the study of pro-opiomelanocortin-derived peptides, but also could be employed to examine the processing pathways of other biologically active polypeptides, in both central and peripheral tissue extracts.