| Popis: |
Human relaxin, a two-chain protein hormone, was synthesized by solid-phase peptide synthesis in combination with a novel thiol-protecting group strategy whereby the three disulfide bonds could be synthesized sequentially and without error. The final product was shown to be homogeneous by reversed-phase high performance liquid chromatography and electrophoresis and had the correct amino acid composition and sequence. Tryptic digestion and peptide mapping of the synthetic relaxin by reversed-phase high performance liquid chromatography resulted in a pattern identical with that produced by standard tryptic relaxin fragments synthetized by different methods. Three human relaxin derivatives containing oxidized methionine, formyltryptophan, and bis[B13,B17-citrulline]-relaxin, were produced and their biological activity and structural similarity to human relaxin was assessed. All derivatives, except those containing modified tryptophan residues, showed indistinguishable circular dichroic spectra, indicating that the modifications did not cause significant structural changes. However, only human relaxin and the tryptophan- and methionine-protected relaxin derivatives showed bioactivity. The derivative in which the two arginines in positions B13 and B17 had been replaced by the uncharged isosteric amino acid citrulline were biologically inactive. This observation confirms preliminary studies (Büllesbach, E. E. and Schwabe, C. (1988) Int. J. Pept. Protein Res. 32, 361-367) that suggested that these two conserved arginines located in the midregion of the relaxin B chain are essential for the function of the hormone. |
