Isolation and physicochemical characterization of human follicle-stimulating hormone isoforms.

Twenty hFSH isoforms were isolated from human pituitary extracts, 15 of which were highly pure. The mild purification procedure, which used a FSH RRA to monitor FSH activity, involved an initial fractionation of pituitary extracts by gel filtration and isoelectric focusing. Six pI regions (mean pI values, 3.63, 3.88, 4.07, 4.23, 4.84, and 5.13) of human (h) FSH were obtained and further fractionated on ion exchange and gel filtration HPLC. Recoveries of FSH radioreceptor activity at each stage were greater than 80%. Fifteen isoform preparations were judged as near homogeneous by HPLC-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibiting elution and migration behaviors consistent with the known mol wt of intact hFSH and its subunits. The remaining 5 isoform preparations contained a higher mol wt component that is probably hFSH related, as this component was detected after iodination, immunoprecipitation with hFSH antiserum, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar amino acid compositions were obtained for the 20 hFSH isoforms, except for evidence of some oxidative degradation of serine, threonine, and tyrosine and decreased levels of glutamic acid, possibly due to carboxy-terminal heterogeneity of the beta-subunit. An average amino acid composition value for all isoforms was comparable to that of 2 other highly purified hFSH preparations. Using the First International Standard for pituitary hFSH (83/575) as standard, radioreceptor activities were obtained ranging from 7,800-56,300 IU/mg protein. It is concluded that a mild purification procedure for the isolation of hFSH isoforms has been developed which gives high recoveries and has enabled the isolation of 15 isoforms in high purity suitable for further physicochemical and biological characterization.