Isolation of inhibin from ovine follicular fluid using reversed-phase liquid chromatography

This report describes the application of high performance liquid chromatography (HPLC) and associated techniques to the isolation of inhibin from ovine follicular fluid (oFF). Two modes of HPLC have been assessed. High speed gel permeation chromatography on a Waters I-125 support showed that at pH 7.0 inhibin bioactivity was associated with an approx. 80000 mol. wt protein fraction which co-eluted with the bulk of the protein in oFF and gave a yield of 30-44% of the applied activity. Reversed-phase HPLC (RP-HPLC) of a peptide-rich fraction extracted from oFF was also investigated. Using analytical liquid chromatography on octadecylsilylsilica (ODS-silica), two distinct inhibin bioactive fractions were obtained, with a resultant purification of 23-fold and 3-fold respectively and an overall recovery of 8.5%. These results demonstrate that RP-HPLC may be employed as a direct and rapid technique for the isolation of inhibin from oFF.