Purification of follicle-stimulating hormone from horse anterior pituitary glands.

This research article is about the process used to purify follicle-stimulating hormone (FSH) from horse anterior pituitary glands and verifying its purity and effectiveness in stimulating estrogen production in immature hypophysectomized rats.

Extraction and Purification Process

• Fresh horse-pituitary glands were first extracted using 40% ethanol. This process helped to retrieve the gonadotropins.
• The concentration of the alcohol was increased up to 85% and was then dried with acetone to further consolidate the extraction.
• The obtained preparation was subjected to water extraction at pH 5. The pH of this extract was then adjusted to pH 7, before undergoing lyophilization, a drying process.
• The FSH in the pH-5-soluble fraction was then purified using zone electrophoresis, a method that separates ions based on their electrokinetic mobility within a homogeneous medium.
• After the electrophoresis, the components were resolved into six distinct parts using starch-gel electrophoresis, another separation method whereby particles migrate in a gel composed of starch, in the presence of an electric field.

Identification and Confirmation of FSH

• Among the six components obtained, one of them contained the follicle-stimulating hormone.
• This FSH was then recovered in the elution cell, a part of the chromatographic system where the separation of substances occur.
• The remaining contaminants, starch in this case, were separated via chromatography on Dowex-2 X8, a type of ion-exchange resin. This ensured the purified FSH was essentially free of other substances.

Verification of the Purified FSH

• The purified FSH stimulated estrogen production in immature hypophysectomized female rats, confirming its functional integrity and effectiveness.
• It was observed to be free from luteinizing hormone and other pituitary hormones which further verified its purity.
• The purified FSH migrated as a single zone in starch-gel electrophoresis, which means there was just one type of particle existing in the solution, i.e., FSH. This was a further testament to the effective purification process.
• It also sedimented as a single boundary in the ultracentrifuge, a high-speed centrifuge, suggesting homogeneity in the sample and hence, purity.