PURIFICATION OF HIGH-MOLECULAR-WEIGHT FOLLICLE-STIMULATING-HORMONE BINDING INHIBITOR IN PORCINE FOLLICULAR FLUIDS

We performed the purification of high-molecular-weight follicle-stimulating hormone binding inhibitor (FSHBI) from porcine follicular fluids. The FSHBI activities of high-molecular-weight fractions acquired by ultrafiltration of follicular fluids from small, medium and large follicles with Centriflo CF25 membrane cone were 277.2 +/- 24.6, 176.7 +/- 3.0 and 141.3 +/- 3.6 U, respectively. By affinity chromatography of CF25 retentate with a column of Blue Sepharose CL6B, 94.1 +/- 5.3% of FSHBI activity was recovered in the unretained fraction. The FSHBI in the unretained fraction was purified by anion-exchange chromatography with a column of Mono Q and gel filtration on Sephacryl S300HR. As a result of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final purified fraction, a single silver-stained band was observed at 310 kD under the non-reducing conditions. On the other hand, under the reducing conditions, SDS-PAGE revealed three bands at 178, 101 and 55 kD. A double reciprocal plot analysis of this substance showed competitive inhibition in FSH binding. The results of the present study suggest the existence of a 310 kD FSHBI composed of three subunits of 178, 101 and 55 kD in porcine follicular fluids.