Pulcherosine, a novel tyrosine-derived, trivalent crosslinking amino acid from the fertilization envelope of sea urchin embryo

Abstract

The normally hardened and aminotriazole-induced soft fertilization envelopes (FEs) of the sea urchin Hemicentrotus pulcherrimus and two other species were isolated and investigated for component proteins and cross-linking amino acids. From the acid hydrolysate of the hard FE of H. pulcherrimus, we isolated by reversed-phase high-performance liquid chromatography a novel fluorescent compound as well as dityrosine and trityrosine, the major tyrosine-derived cross-linking amino acids. These three compounds were also isolated from the reaction products of the tyrosine/horseradish peroxidase/H2O2 system. The structure of the novel compound, designated "pulcherosine", was determined to be 5-[4"-(2-carboxy-2-aminoethyl)phenyoxy]-3,3''-dityrosine. With respect to the position of diphenyl ether bond between the tyrosine and dityrosine moieties, it is an isomer of isotrityrosine found in Ascaris cuticle collagen [Fujimoto et al. (1981) Biochem. Biophys. Res. Commun. 99, 637-643]. Isotrityrosine was not found in either of the above systems as a major component. The contents of tyrosine, dityrosine, trityrosine, and pulcherosine in the hard FE of H. pulcherrimus were estimated as 255, 5.5, 2.1, and 1.3 residues, respectively, per 10000 total amino acid residues, while in the soft FE, those of tyrosine and dityrosine were 305 and 0.25 residues, respectively, and trityrosine and pulcherosine were only traces. The molar ratio of dityrosine, trityrosine, and pulcherosine in the hard FE was 100:38:24, while that for tyrosine/horseradish peroxidase/H2O2 reaction products was 100:3:8, respectively.