Facile separation of the subunits of ovine and bovine gonadotrophins by high-performance liquid chromatography: microheterogeneity and biological activity.
The alpha and beta subunits of ovine and bovine gonadotrophins which undergo instant dissociation in 0.1% (v/v) trifluoroacetic acid in water could be separated into their multiple components by reverse-phase high-performance liquid chromatography on analytical and preparative scales. Several batches of highly purified ovine LH (oLH) separated into four alpha components (alpha 1 to alpha 4) and five beta components (beta 1 to beta 5). While the major alpha component (alpha 3) remained the same in all, the beta subunit was variable in all these preparations of oLH of equal biological and immunoreactivity. Various manipulations, such as prolonged incubation in dilute acid or alkali or conditions prone to air oxidation, did not change elution patterns or produce interconversions among the alpha or beta components, suggesting that microheterogeneity arose in the pituitary itself or during its collection. The subunits obtained on a preparative scale were of high yield and quality and recombined (100%) to produce fully active LH or FSH. Chemical deglycosylations in which 75-80% of the peripheral sugars were removed shifted the elution of all components (alpha as well as beta) slightly but modifications to the protein backbone by other reagents changed the profile of the alpha subunit. Obtaining the various alpha and beta components in high yield would permit a complete analysis of gonadotrophin microheterogeneity.
Sairam
M R
MR
Reproduction Research Laboratory, Clinical Research Institute of Montreal, Québec, Canada.
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005640:Follicle Stimulating Hormone / Q000032:analysis* / Q000737:chemistry; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit / Q000032:analysis* / Q000737:chemistry; D007211:Indoles / Q000494:pharmacology*; D007986:Luteinizing Hormone / Q000032:analysis* / Q000737:chemistry; D012756:Sheep
D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D007211:Indoles; C071554:ZK 119010; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone