Porcine Inhibin: Initial Fractionation as a High Molecular Weight Complex

Abstract

A high molecular weight complex or aggregate of inhibin was obtained by chromatography of porcine follicular fluid in Fractogel TSK65F. Recovery of activity was good (usually 80-100%), but only 30-60% was recovered as a high molecular weight complex (greater than 160,000) free of albumin and gamma globulin (the two major proteins in follicular fluid). The balance of the activity was distributed in the gamma globulin-albumin region of the chromatogram (i.e., 160,000 down to 65,000 daltons). Distribution in this region of the chromatogram in part reflected the prior processing of the sample (e.g., it was augmented by ethanol or acetone precipitation prior to chromatography). The utility of Fractogel chromatography lies in its ability to resolve a large portion of the inhibin activity from the major proteins (albumin and gamma globulin), plus an efficient recovery of activity.

Maximum purification on the Fractogel chromatograms was approximately 20-fold. This product and the other Fractogel fractions were tested for protease activity by a sensitive slab gel procedure. All fractions contained detectable protease activity that could potentially affect inhibin activity during further fractionations. This was shown with a protease fraction isolated from porcine follicular fluid by affinity chromatography. When added to a partially purified inhibin preparation, this protease fraction destroyed 77% of the inhibin activity.