Determination and occurrence of tyrosine O-sulfate in proteins

Abstract

This chapter focuses on the detection of tyrosine-sulfated proteins and on the determination of tyrosine sulfate by methods that are based on labeling proteins with 35SO4. This approach has several advantages over the chemical determination of tyrosine sulfate in unlabeled proteins. It can be much more easily used to search for tyrosine-sulfated proteins and is considerably more sensitive, and therefore requires less protein material for analysis. The chapter discusses an immunological approach to detect tyrosine-sulfated proteins. In this approach, antisera were raised against a synthetic antigen containing a large number of tyrosine sulfate residues and antibodies were purified from the antisera by affinity chromatography. These antibodies appeared to recognize tyrosine sulfate-containing proteins in “Western” blots and in solid-phase radioimmunoassay. The general usefulness of these antibodies to screen for, immunoprecipitate, or purify tyrosine-sulfated proteins is currently being tested. The standard method to detect tyrosine sulfate in proteins involves four main steps: (1) in vivo labeling of tissues in situ or of tissue explants, tissue slices, and cells in culture with inorganic [35S]sulfate; (2) the separation of proteins by polyacrylamide gel electrophoresis (PAGE); (3) elution from gels and the hydrolysis of individual 35SO4-1abeled proteins; and (4) the separation of tyrosine [35S]sulfate by thin-layer electrophoresis.