Isolation and partial characterization of prolactin from equine pituitary gland (hypophysis).

Researchers have successfully isolated and partially characterized prolactin from the pituitary gland of a horse, through a series of extraction and purification processes. The purified hormones showed a biological potency and were verified using various testing methods, providing new insights into equine hormone activity.

Methodology and Process of Isolation

• In this study, the pituitary gland of a horse, also known as the hypophysis, was obtained and its prolactin hormone was extracted. This involved a series of serial extractions with different solvents at varying pH levels to remove other hormones and impurities.
• Extraction was initiated with water at pH 5.5, then using 0.1 M ammonium sulphate at pH 4.0, and later, 0.25 M ammonium sulphate at pH 5.5. The aim was to get rid of undesirable components.
• After other hormones were removed, prolactin was then extracted using 70% ethanol at a pH level ranging from 9.3 to 10.0.
• Preliminary purification of the extracted prolactin involved the use of 0.1% sodium chloride at a pH of 9.0 to salt out other substances.

Purification and Testing

• Following preliminary purification, prolactin was precipitated out by adding thrice the volume of 95% ethanol at 4 degrees Celsius.
• The resulting prolactin preparation showed a biological potency of 24 International Units (IU) per milligram.
• Further purification was done by isoelectric focusing on a pH gradient of 5 to 7, which gave three different prolactin components.
• These components had isoelectric points at 5.8, 5.7, and 5.25 and biological potencies (in IU/mg) of 35.6, 19.6, and 11.3 respectively.

Characteristics and Reactions

• The major component isolated had a molecular weight of 25,000 and showed the highest isoelectric point and biological potency.
• An antiserum was produced against this major component for further investigations.
• This antiserum did not cross-react with equine follicular stimulating hormone, luteinizing hormone, and growth hormone, indicating the specificity of the antiserum for equine prolactin.
• The antiserum was found to cross-react with prolactin from sheep (ovine) and cattle (bovine), although human and mice (murine) prolactins showed little cross-reactivity with the equine prolactin antiserum. This suggests some similarities and differences in the structures of prolactin across different species.