Development of a novel reagent for radiolabeling of peptides and proteins

Objectives: Radiolabeling of a tyrosine residue in peptides and proteins is routinely performed by using various oxidizing agents such as Chloramine T, Iodobeads, and Iodogen reagent and radioactive iodide (I^-). The objective of the present study was to develop a novel reagent for radiolabeling of peptides and proteins.

Methods: An inorganic monochloramine (NH₂Cl) was investigated as a novel reagent for radiolabeling of a tyrosine residue in peptides and proteins. A known amount of carrier-free ¹²⁵I Na activity was added to a 100 µL of 0.1 M Sodium Phosphate (pH 7.4) buffer solution containing a known amount of tyrosine amino acid or a cyclic peptide (cRGDyK, cyclo Arg-Gly-Asp-d-Tyr-Lys) or a protein (BSA, Bovine Serum Albumin) in a 1.5 mL Eppendorf centrifuge tube. Excess NH₂Cl in PBS buffer was added next and the reaction mixture was incubated at room temperature for 20 minutes. Excess NH₂Cl was reduced by the addition of sodium metabisulfite. The crude reaction mixture was purified by using either a Sep-PaK C₁₈ Light cartridge or a PD-10 column. The final product was analyzed by a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) analytical method.

Results: The RP-HPLC and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectral analysis of cold iodinated Tyrosine and cRGDyK suggested the formation of mono and diiodinated Tyrosine and cRGDyK. Radiolabeling efficiency (i.e., incorporation of ¹²⁵I label) were: 100% for Tyrosine, 86% for cRGDyK, and 82% for BSA. Reversed-Phase (for radiolabeled Tyrosine and cRGDyK) and Size-Exclusion (for radiolabeled BSA) HPLC analysis showed >99% Radiochemical Purity (RCP) of the radiolabeled materials.

Conclusion: A novel reagent for radiolabeling of peptides and proteins was developed successfully. Research Support: