Identification of tyrosine O-sulfate in proteins by reverse-phase high-performance liquid chromatography: Use of base hydrolysis combined with precolumn derivatization using phenyl isothiocyanate

Abstract

A procedure has been developed for the analysis of tyrosine O-sulfate in proteins. Samples are subjected to base hydrolysis with Ba(OH) 2, neutralized with sulfuric acid, and the majority of other amino acids removed by chromatography on Dowex AG 50 × 8. The average recovery of tyrosine O-sulfate from these procedures was 43%. Tyrosine O-sulfate was identified by reversephase HPLC as the phenylthiocarbamyl derivative following precolumn derivatization with phenyl isothiocyanate. The method has been applied to bovine fibrinogen giving a tyrosine O-sulfate content ranging from 0.59 to 1.23 mol/mol. These procedures were also shown to be suitable for the analysis of the incorporation of [ 35S]sulfate into tyrosine O-sulfate residues in proteins by intact cells.