Affinity purification and sequence determination of equine relaxin.

This study explores a more efficient method to purify relaxin, a hormone often associated with pregnancy, from horses. Through affinity chromatography paired with high-performance liquid chromatography (HPLC), four main and several minor versions (isoforms) of equine relaxin were isolated and studied.

Methodology

• The researchers raised monoclonal antibodies against highly purified equine relaxin. They produced large quantities of the antibodies by inducing ascites, a condition where fluid builds up in the abdomen.
• The antibodies were tied to a solid physical support, creating an affinity column, through which a blend of term equine placentas was passed. The relaxin was isolated by altering the pH, causing it to detach from the antibodies and be collected.
• Different isoforms of equine relaxin were separated by HPLC with the use of a C18 (ODS) reverse phase column and a linear gradient of 25-30% acetonitrile.

Results

• Four main and a few minor isoforms of equine relaxin were obtained. These isoforms demonstrated similar molecular weights, were composed of subunits, and displayed similar charges.
• Positivity for biological activity was proven in five isoforms using the mouse interpubic ligament bioassay.
• The researchers sequenced equine relaxin using Edman degradation, a process for sequencing proteins by successively removing one amino acid at a time. Verification was done through fast atom bombardment mass spectrometry, a method for determining the molecular weight and sequence of a substance.
• The differences between the isoforms of equine relaxin were found to be based on variations at the C-terminus of the B-chain, a specific region of the protein.

Conclusions

• Equine relaxin appears to be the smallest sequenced relaxin. It contains an A-chain with 20 residues and a B-chain with 28 residues, giving it a total molecular weight of 5253.
• The greatest sequence homology, or similarity, of equine relaxin was found to be with the relaxin from pigs, exhibiting a 67% identity. This highlights the evolutionary connection between these species and suggests potential functional similarities in their relaxin proteins.