Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.
Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.
Mathieu
M N
MN
Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Australia. m.mathieu@hfi.unimelb.edu.au
Wade
J D
JD
Catimel
B
B
Bond
C P
CP
Nice
E C
EC
Summers
R J
RJ
Otvos
L
L
Tregear
G W
GW
D000818:Animals; D015374:Biosensing Techniques; D001710:Biotin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D011487:Protein Conformation; D051381:Rats; D012065:Relaxin / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001710:Biotin; D012065:Relaxin