Proceedings: Techniques for the purification of human pituitary follicle-stimulating hormone.

The glycoprotein hormones of the anterior pituitary and of the placenta are made up of a and p subunits, one of which, the a, is common to each hormone. Thcy therefore share some similar physicochemical and immunological properties, and the complete purification of follicle-stimulating hormone, for instance, has proved difficult. Many preparations which have appeared to be pure by one criterion have shown heterogeneity by another. Thus bioassay may reveal contaminating luteinizing hormone or specific radioimmunoassay may detect either luteinizing hormone or thyroid-stimulating hormone. Further, many of the apparently pure preparations described show a wide range of biological activities. Loss of biological activity may occur during storage or during fractionation and concentration procedures. This may be the result of dissociation into subunits or of small chemical modifications. Thus activity decreases sharply with the loss of sialic acid residues, which occur at the terminal non-reducing ends of the carbohydrate chains (Butt, 1970; Kennedy & Butt, 1969). These residues are labile and are readily cleaved at low pH. They carry considerable charge and forms of the hormone with variable amounts of sialic acid may be separated by isoelectric focusing. Luteinizing hormone is the most difficult of the glycoprotein hormones to remove from follicle-stimulating hormone : the separation is usually achieved by making use of their different isoelectric points in the techniques of electrophoresis or ion-exchange chromatography. Luteinizing hormone contains less sialic acid than follicle-stimulating hormone and the complete separation of these two may be made more difficult when some of the sialic acid residues have been removed from follicle-stimulating hormone. One of the most successful methods has been described by Roos(1968), whoworked on fresh frozen glands. They were extracted in a solution of phosphate buffer and purification proceeded by fractionation with (NH4)2S04, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, chromatography on hydroxyapatite and electrophoresis on polyacrylamide gel. Organic solvents were avoided throughout and concentration in between columns was by ultrafiltration. The final product was not dried because there was some evidence that as purification proceeds the stability of the hormone decreases, and there was danger that loss of biological activity may have resulted from such a process as freeze-drying. A rather similar method was described for acetone-dried glands by Peckham & Parlow (1969). In several other methods the initial extraction from acetone-dried glands has been by use of mixtures of ammonium acetate and ethanol with the subsequent steps similar to those of Roos (1968) (Reichert et al., 1968; Saxena & Rathnam, 1971 ; Butt & Lynch, 1972). The method we have used consists of extraction of the acetone-dried glands in ammonium acetate-ethanol (3 :2, v/v; pH6.1) followed by chromatography on CMcellulose to separate the major portion of follicle-stimulating hormone from luteinizing hormone and thyroid stimulating hormone (Hartree, 1966). The extract was precipitated from ethanol, redissolved in 1 mM-phosphate buffer at pH 7.5 and submitted to fractionation on calcium phosphate and DEAE-cellulose, and finally to gel filtration on Sephadex G-100 to separate subunit material. The chromatography on DEAE-cellulose is particularly important, since it is at this stage that most of theresidual luteinizinghormoneis removed. The process is best monitored by radioimmunoassays : most of the contaminating luteinizing hormone is eluted in 0.075~-ammonium acetate and the remainder with follicle-stimulating hormone on increasing the molarity to 0 .125~ . The latter is eluted from the Sephadex column in a position corresponding to a Stokes’ radius of 3.1 nm. The biological activity averages about SO00 i.u./mg and the contamination due to luteinizing hormone is about 150 i.u./mg as measured by radioimmunoassay. Similar