Synthesis, conformational studies and biological activity of N-alpha-mono-biotinylated rat relaxin

Biotin-avidin immobilization can be a useful tool in structure-function stu dies of hormone receptors. A crucial step is the preparation of a specifica lly biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxi n, 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 conve nient analogue because reliable bioassays exist, thus allowing assessment o f the effect of N-biotinylation on bioactivity. To help improve the yield o f the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzy l (H mb) backbone protection was used during the solid-phase assembly of th e 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 in troduced at the N-terminus of the B-chain using standard coupling protocols . The chain combination with the A-chain was accomplished in reasonable yie ld. Secondary structural measurements demonstrated that the biotin caused t he starting B-chain to adopt a more ordered conformation. The labelled synt hetic relaxin exhibited similar circular dichroism spectra to native and sy nthetic single B-chain peptides. In addition, the biotinylated relaxin show ed no significant difference in its chronotropic activity in the rat isolat ed heart assay compared with the native peptide. Biosensor studies showed t hat antibody recognition was retained upon attachment of the synthetic rela xin to the streptavidin-derivatized surface.