Amino Acid Composition and Molecular Weight of Human Follicle Stimulating Hormone 1

Abstract

Conflicting reports in regard to the biological potency of purified human pituitary follicle stimulating hormone (FSH) preparations have appeared in the literature (1-4). The potencies of such preparations have ranged from 45 to about 500 times NIH-FSH-SI. Reports on the chemical composition of FSH similarly contain significant discrepancies (1-5). Studies of human FSH, therefore, continue to be of interest.

We have obtained a homogenous human FSH preparation, by employing a series of simple column fractionation steps, and have examined some of its physicochemical characteristics.

Methods, Results and Discussion. Acetonedried human pituitary powder was extracted and purified according to the procedure of Hartree (6). The crude material was further purified on a column of DEAE-C (2), followed by fractionation on Sephadex G-100, and then rechromatography on DEAE-Sephadex (4). Final purification was effected by absorption onto sulfoethyl Sephadex (7). Inactive material was removed by stepwise elution with acetate buffer, ph 4, ionic strength 0.03, and phosphate buffer, pH 6, ionic strength 0.03. FSH eluted with 0.01 M NA₂ HPO₄, was exhaustively dialyzed against distilled water at 4° and stored frozen at —70°.

Biological assay for FSH was performed by the method of Steelman and Pohley (8), as modified (9). Biological assay for LH was performed by the method of Parlow (10). All solutions used for bioassay were thawed and appropriately diluted immediately prior to injection. The dosage was based on the quantity of FSH determined either by the Lowry and co-workers'method (11) or by obtaining the dry weight of the freshly lyophilized material. The value from the former procedure corresponds to 78% of the dry weight (12).

The purified FSH has a biologic potency of 120X NIH-FSH-S1/mg. LH contamination was 0.24X NIH-LH-S1. The FSH preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis, in which it moved as a single broad band at pH 8.3 (Fig. 1).