Inhibin fractionation: a comparison of human and porcine follicular fluid, with particular reference to protease activation.
A novel treatment has been devised in our studies of the purification of inhibin from porcine and human follicular fluids (pFFl and hFFl, respectively). Both pFFl and hFFl were precipitated with acetone and extracted with acetic acid to provide a starting material for subsequent gel filtration and reverse-phase high-pressure liquid chromatography (HPLC). Inhibin from pFFl was purified 4200-fold using this methodology. Inhibin from hFFl could not be purified to this degree since recoveries were relatively poorer than for pFFl and yielded too little material for the HPLC step. In our fractionation scheme, protease activities were assessed with a gel electrophoresis assay system. Protease activity at approximately 90 kDa was observed in raw pFFl. When inhibin was fractionated by extraction or chromatography, additional bands of protease activity appeared near 150 kDa, 66 kDa and at less than 45 kDa. In raw hFFl, only faint bands of protease activity were observed at approximately 90 kDa and at 85-90 kDa. Upon further fractionation of hFFl, protease activity was reduced below the ability of this method to detect it. Our results suggest that, with our treatment of follicular fluid, protease activity is present in pFFl and additional protease activity appears upon fractionation; proteases, although present, do not eliminate the possibility of obtaining a highly purified inhibin preparation with acceptable recoveries of inhibin activity during purification; and although protease activity could be reduced or eliminated from hFFl, the low yields of inhibin activity from this method mandate a different approach to purification of inhibin from hFFl.
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006801:Humans; D007265:Inhibins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006080:Ovarian Follicle / Q000032:analysis / Q000201:enzymology / Q000378:metabolism; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism; D013045:Species Specificity; D013552:Swine
D007265:Inhibins; D005640:Follicle Stimulating Hormone; D010447:Peptide Hydrolases