Inhibin isolated from ovarian follicular fluid and method for the isolation and purification of inhibin

摘要

A protein which satisfies all the biological criteria which are characteristic for inhibin has been isolated form a gonadal source. The purification and characterisation of inhibin and the use of the purified material for forming antibodies, the use of inhibin and antisera in a quantitative radioimmunoassay, and in vitro and in vivo uses of inhibin and antibody against inhibin. A purified protein, i.e. inhibin is provided, which protein is characterized in that (a) the apparent molecular weight as determined by SDS- PAGE is 56,000 +- 1,000, (b) the isoelectric point is in the range of 6.9-7.3, (c) the protein can bind specifically to Concanavalin A- Sepharose, (d) the protein consists of two subunits which can be separated after reduction, as determined by SDS-PAGE, characterized in that (i) their apparent molecular weights are 44,000 +- 3,000 and 14,000 +- 2,000, respectively, (ii) the isoelectric point for the subunit having the molecular weight of 44,000 is in the range 6.0-7.0, (iii)the N-terminal amino acid sequences of the two subunits are as described in this publication, (e) the protein can suppress follicle stimulating hormone but not luteinising hormone, thyroid stimulating hormone or prolactin in an in vitro bioassay system, (f) the protein can be labelled with radioactive iodine. A process is also provided for isolating and purifying inhibin from material of mammalian origin, characterized in that the starting material is subjected to the following steps: (a) one or more gel permeation chromatography steps, (b) one or more HPLC chromatography steps using reverse phase, (c) one or more preparative polyacrylamide gel electrophoresis steps, (d) electrophoretic elution of the purified inhibin.