2. School of Chemistry, University of Melbourne, Victoria 3010, Australia.
3. Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia.
4. Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia
Background: The chemical synthesis of multi-disulfide bonded heterodimeric peptides such as insulin has long been of significant scientific and commercial interest as well as a major challenge. The development of improved protocols which includes regioselective disulfide bond formation has greatly advanced the capacity to prepare and study insulin-like peptides including canine relaxin, an important regulator of parturition and indicator of canine maternal health.
Methods: Separate, efficient solid phase synthesis of the two constituent chains (24 residue A and 35 residue B) was followed by stepwise formation of each of the three disulfide bonds, one intra within the A-chain and two interchain, by oxidation, thiolysis and iodolysis respectively.
Results: Synthetic canine relaxin having a total of 59 residues was prepared in good overall yield and shown by several criteria to be highly purified. The peptide was shown to be less potent than human relaxin (H2 relaxin) in binding to and activating the human relaxin receptor, RXFP1, in transfected cells. A circular dichrosim spectroscopic analysis showed that the canine relaxin also possessed significantly less secondary structure compared to H2 relaxin which may account for its reduced activity.
Conclusions: The synthetic protocols developed in our laboratory enabled the successful preparation of the complex, small insulinlike protein, canine relaxin. This will, in turn, allow a detailed study of both the tertiary conformation of this peptide and its role in canine reproduction.
Keywords: Canine relaxin, H2 relaxin, circular dichroism spectroscopy, regioselective disulfide bond formation, RXFP1 receptor assay, solid phase peptide synthesis