Structural studies on equine glycoprotein hormones. Amino acid sequence of equine lutropin beta-subunit.

This study identified the amino acid sequence of equine lutropin beta (eLH beta), a glycoprotein hormone in horses, through various methods including digestion with V8 protease, cyanogen bromide cleavage, and acid-labile Asp-Pro bond cleavage. The study found that eLH beta’s amino acid sequence is identical to that of equine chorionic gonadotropin beta.

Experiment Procedures

• The amino acid sequence of equine lutropin beta (eLH beta), a glycoprotein hormone in horses, was determined via a multi-step process.
• At first, large fragments of eLH beta were obtained via several chemical and enzymatic methods – digestion with Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, and breaking down of acid-labile Asp-Pro bonds.
• The resulting fragments were subsequently purified through processes such as gel filtration and high performance liquid chromatography (HPLC).
• Automated Edman degradation, a sequencing method for proteins, was used to sequence the eLH beta. Some peptides went through further digestion with the enzyme chymotrypsin for additional purification via HPLC.

Key Findings

• Both automated Edman degradation and the complementary 5-dimethylaminonaphthalene-1-sulfonyl-Edman procedure were used to sequence the more purified peptides.
• The researchers were able to identify glycosylated amino acids directly through the aforementioned complementary procedure.
• eLH beta was found to be a glycoprotein composed of 149 amino acids, which consist of both N- and O-linked oligosaccharides.
• Interestingly, the sequence also possesses a COOH-terminal extension that is similar to the one present in human chorionic gonadotropin, another type of hormone.
• This COOH terminus appears to be blocked by glycosylation, as suggested by the results of carboxypeptidase Y digestions.
• The sequence of amino acids in eLH beta was revealed to be identical to that in equine chorionic gonadotropin beta, a significant discovery in the study.