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.