Isolation and structural determination of a novel TRH-like tripeptide, pyroGlu-Tyr-Pro amide, from alfalfa.

Abstract

The tripeptide pyroGlu-Tyr-Pro amide was isolated from an aqueous extract prepared from dried alfalfa pellets. The tripeptide was quantitated using a competitive radioimmunoassay in which 125I-labeled thyrotropin-releasing hormone (TRH), is displaced from antibody specific to TRH (pyroGlu-His-Pro amide). The pyroGlu-Tyr-Pro amide was purified by passing the filtered extract through QAE-Sephadex A25 at pH 5, followed by open bed chromatography on C18 silica using an H2O/methanol gradient, then preparative high performance liquid chromatography (HPLC) on microbondapak C18 using a 10 mM HCl/methanol gradient, followed by G-10-Sephadex chromatography, SP-C25-Sephadex chromatography, QAE-Sephadex chromatography at pH 10.1, analytical HPLC on a microbondapak C18 column eluted with 10 mm HCl/acetonitrile, and analytical HPLC reverse phase chromatography on an APEX phenyl column eluted with H2O/acetonitrile. The tripeptide was essentially homogeneous after the final chromatography step, as judged by correspondence of immunoreactivity with A280. The sequence of the alfalfa tripeptide was determined to be Glu-Tyr-Pro by gas phase sequencing, after hydrolysis of pyroglutamic acid by mild acid hydrolysis. The mass of the alfalfa tripeptide was 389.1, as determined by fast ion bombardment mass spectroscopy, and was found to be identical to the mass of synthetic pyroGlu-Tyr-Pro amide. The sequence of the alfalfa tripeptide was also verified using B/E-linked scanning. I conclude that the tripeptide isolated from alfalfa differs from human thyrotropin-releasing hormone only by the substitution of tyrosine for histidine at position 2. The role of pyroGlu-Tyr-Pro amide in alfalfa is not known, but the existence of a family of thyrotropin-related peptides occurring in both the animal and the plant kingdoms indicates that the thyrotropin related peptides have a wide phylogenetic distribution.