Regioselective Disulfide Solid Phase Synthesis, Chemical Characterization and In Vitro Receptor Binding Activity of Equine Relaxin

  • Mohammed Akhter HossainEmail author
  • Feng Lin
  • Soude Zhang
  • Tania Ferraro
  • Ross A. Bathgate
  • Geoffrey W. Tregear
  • John D. Wade


In the equine industry, pregnancy loss during the third trimester constitutes a large percentage of fetal and neonatal mortality and represents a major financial loss and time investment for the breeder. Early identification of placental insufficiency would, in some cases, make it possible to sustain the pregnancy through medical intervention. Recent work suggests that relaxin is a valuable clinical tool for diagnosing placental insufficiency and monitoring treatment efficacy in mares. Relaxin is a polypeptide member of the insulin superfamily that consists of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. It is typically produced in the ovary during pregnancy and has primary roles in maintaining mammalian pregnancy and facilitating the delivery of the young via remodelling of the reproductive tract. The placenta is the primary source of relaxin in the mare during pregnancy. Its primary structure has been determined and shown to be the smallest of the known mammalian relaxins. It consists of a 20 residue A-chain and a 28-residue B-chain. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken using regioselective disulfide formation methods. The synthetic equine relaxin showed typical α-helical structure under physiological conditions. The peptide was found to bind to the relaxin receptor, LGR7, in vitro, and its binding affinity was found to be higher than that of the “gold standard”, porcine relaxin, and similar to that of the human relaxin-2 (H2 relaxin).


Circular dichroism spectra equine relaxin regioselective disulfide bond formation solid phase peptide synthesis 



The work described herein was supported by an institute block grant (reg key #983001) and project grant to JDW (#350284) from the NHMRC of Australia.


  1. Akaji K., Fujino K., Tatsumi T., Kiso Y. (1993), J. Am. Chem. Soc. 115:11384–11392CrossRefGoogle Scholar
  2. Atherton E., Sheppard R. C. (1998). Solid-Phase Synthesis: A Practical Approach. IRL press, OxfordGoogle Scholar
  3. Bathgate , R. A. D., Lin, F., Hanson, N. F., Otvos, Jr. L., Guidolin, A., Giannakis, C., Bastiras, S., Layfield, S., Ferraro, T., Ma, S., Zhao. C., Gundlach, A. L., Samuel C. S., Tregear, G. W. and Wade, J. D.: 2005, Biochemistry 45, 1043–1053 CrossRefGoogle Scholar
  4. Büllesbach E. E., Schwabe C. (1991), J. Biol. Chem. 266: 10754–10761PubMedGoogle Scholar
  5. Büllesbach E. E., Schwabe C. (1995) Intl. J. Pept. Prot. Res. 46, 238–243CrossRefGoogle Scholar
  6. Büllesbach E. E., Schwabe C. (2001) Eur. J. Biochem. 241: 533–537CrossRefGoogle Scholar
  7. Dawson N. F., Tan Y. Y., Macris M., Otvos L., Summers R. J., Tregear G. W., Wade J. D. (1999). J. Pept. Res. 53: 542–547CrossRefPubMedGoogle Scholar
  8. Klonisch T., Mathias S., Cambridge G., Hombach-Klonisch S., Ryan P. L., Allen W. R. (1997). Placenta 18: 121–128CrossRefPubMedGoogle Scholar
  9. Lin, F., Otvos, Jr. L., Kumagai, J., Tregear, J. W., Bathgate, R. A. D. and Wade, J. D.: 2004, J. Pept. Sci. 10, 257–264.CrossRefPubMedGoogle Scholar
  10. Luo P., Baldwin R. L. (1997). Biochemistry 36: 8413–8421CrossRefPubMedGoogle Scholar
  11. Maruyama K., Nagasawa H., Isogai A., Ishizaki H., Suzuki A.: (1992a). J. Protein Chem. 11: 1–12CrossRefGoogle Scholar
  12. Maruyama K., Nagasawa H., Isogai A., Ishizaki H., Suzuki A.: (1992b). J. Protein Chem. 11: 13–20CrossRefGoogle Scholar
  13. Maruyama, K., Nagasawa, H. and Suzuki, A.: 1999, Peptides 20, 881–884CrossRefPubMedGoogle Scholar
  14. Ryan, P. L., Vaala, W. E. and Bagnell, C. A.: (1998). American Association of Equine Practitioners Proceedings 62–63Google Scholar
  15. Ryan P. L., Bennet-Wimbush K., Vaala W. E., Bagnell C. A. (1999). Pferdeheilkunde 15: 622–626Google Scholar
  16. Ryan P. L., Bennet-Wimbush K., Vaala W. E., Bagnell C. A. (2001). Therionology 56: 471–483CrossRefGoogle Scholar
  17. Scholtz J. M., Qian H., York E. J., Stewart J. M., Baldwin R. L. (1991). Biopolymers 31: 1463–1470CrossRefPubMedGoogle Scholar
  18. Smith K. J., Wade J. D., Classz A. A., Otvos Jr. L., Temelcos C., Kubota Y., Hutson J. M., Tregear G. W., Bathgate R. A. (2001). J. Pept. Sci. 7: 495–501CrossRefPubMedGoogle Scholar
  19. Sudo S., Kumagai J., Nishi S., Layfield S., Ferraro T., Bathgate R. A., Hsueh A. J. (2003). J. Biol. Chem. 278: 7855–7862CrossRefPubMedGoogle Scholar
  20. Stewart D. R., Stabenfeldt G. H. (1981). Biol. Reprod. 25: 281–289CrossRefPubMedGoogle Scholar
  21. Stewart D. R., Stabenfeldt G. H., Hughes J. P., Meagher D. M. (1982). Biol. Reprod. 27: 17–24CrossRefPubMedGoogle Scholar
  22. Stewart D. R., Papkoff H. (1986). Endocrinology 119: 1093–1099PubMedGoogle Scholar
  23. Stewart D. R., Papkoff H. (1991). Endocrinology 129: 375–383PubMedCrossRefGoogle Scholar
  24. Stewart D. R., Addiego L. A., Pascoe D. R., Haluska G. J., Pashen R. (1992). Biol. Reprod. 46: 648–652CrossRefPubMedGoogle Scholar
  25. Tang J., Wang Z., Tregear G. W., Wade J. D. (2003). Biochemistry 42: 2731–2739CrossRefPubMedGoogle Scholar
  26. Wade, J. D. and Tregear, G. W.: Meths. Enzymol. 1997, 289, 637–646CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Mohammed Akhter Hossain
    • 1
    • 2
    Email author
  • Feng Lin
    • 1
  • Soude Zhang
    • 1
  • Tania Ferraro
    • 1
  • Ross A. Bathgate
    • 1
  • Geoffrey W. Tregear
    • 1
  • John D. Wade
    • 1
  1. 1.Howard Florey Institute of Experimental Physiology and MedicineUniversity of MelbourneParkvilleAustralia
  2. 2.Howard Florey Institute of Experimental Physiology and MedicineUniversity of MelbourneParkvilleAustralia

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