The combined use of size-exclusion and reversed-phase high-performance liquid chromatography for characterization of...

A method is described for the separation and analysis of multiple molecular forms of immunoreactive beta-endorphin and its alpha-N-acetylated congeners by a combination of reversed-phase and size-exclusion high-performance liquid chromatography coupled with two specific radioimmunoassays. Both chromatographic procedures are fast (less than 50 min per analysis) providing good resolution and high recovery (greater than 90%). The solvents used in both systems are ultraviolet transparent (less than 214 nm), non-corrosive, low salt (less than 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 beta-endorphin and alpha-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.