tyrosine-o-sulfate and gastrin-17

Sulfation is a potentially important post-translational modification of proteins and has been demonstrated in a number of polypeptides, notably in gastrointestinal hormones. In contrast to phosphorylation, however, the investigation of sulfation patterns in tissues and on purified proteins has been complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO(3)H) antibody) using CE and a panel of sulfated and nonsulfated peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide (CCK8) in the 1-3 microM range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a K(d1) of 20.1 microM for the monovalent complex. The same binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody-antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material. Furthermore the specificity of this antibody could be confirmed in one operation using an array of sulfated and nonsulfated compounds.

Topics: Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Complement C4; Electrophoresis, Capillary; Gastrins; Hirudins; Immunoglobulin G; Peptide Fragments; Protein Binding; Sincalide; Surface Plasmon Resonance; Tyrosine

Tyrosine O-sulfation is a posttranslational modification of secretory and membrane proteins transported through the Golgi apparatus, which is widespread among higher eukaryotes. O-Sulfated tyrosines are not immediately identified during sequencing of peptides and proteins, because the sulfate ester is acid labile and rapidly hydrolyses to tyrosine in strong acidic solutions. Little is known about the hydrolysis at mildly acidic solutions, which are used during several protein purification and analysis procedures. We have examined the stability of tyrosine sulfate using sulfated gastrin-17, caerulein, and drosulfokinin as models for tyrosine O-sulfated peptides. The peptides were incubated in acidic solutions in a pH range of 1 to 3 at different temperatures and time spans. Only marginal hydrolysis of gastrin-17 was observed in triflouroacetic acid at room temperature or below. Comparison of the acid hydrolysis of the three peptides showed that hydrolysis rate depends mainly on the primary amino acid composition of the peptide. The activation energy (E(a)) for the hydrolysis of sulfated gastrin-17 was found to be E(a)=98.7+/-5 kJ mol(-1). This study serves as a general reference for handling tyrosine sulfated peptides in aqueous acidic solutions. We conclude that tyrosine sulfate is more stable under normal protein purification conditions than previously assumed.

Topics: Ceruletide; Gastrins; Hydrogen-Ion Concentration; Hydrolysis; Neuropeptides; Oligopeptides; Peptide Fragments; Protein Processing, Post-Translational; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Time Factors; Tyrosine