title: Lysine-directed radioiodination of proteins with a cyanuric chloride derivative of aminofluorescein.
abstract: Protein radioiodination via iodo-5-([4,6-dichlorotria- zin-2-yl]amino)fluorescein(DTAF), a cyanuric chloride derivative of aminofluorescein, was characterized. Commercially available DTAF was iodinated by the Iodogen reaction and then conjugated to IgC antibodies in a 4-h incubation in borate buffer, pH 9.0. With low amounts of protein, 10 micron, molar ratios of iodine:IgC of nearly 1:1 were obtained. With 25 micron protein, which was used routinely, 15-20% efficiency of (125)I incorporation was obtained. Polylysine was labeled efficiently, and labeling was inhibited by ethanolamine, which is consistent with previous data indicating that conjugation is to amino in the protein. A low level of aggregates, primarily dimers, was generated. This procedure is a simple, inexpensive method to test the advantage of lysine-directed protein iodination, with proteins for which oxidative iodination is unsuitable. Abs labeled with DTAF retained strong antigen-binding activity, and, in the case of one Ab, were much more active than the chloramine T-labeled Ab. Catabolic products of iodo-DTAF conjugates, produced after internalization and degradation of Ab bound to the cell surface, were retained within some but not all human carcinoma cell lines.
authors.affiliation: Center for Molecular Medicine and Immunology, Garden State Cancer Center, Newark, New Jersey 07103, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Harapanhalli
authors.forename: W
authors.initials: W
authors.lastname: Schafranek
authors.forename: G L
authors.initials: GL
authors.lastname: Ong
authors.forename: M J
authors.initials: MJ
authors.lastname: Mattes
mesh_terms: D019793:Fluorescein; D005452:Fluoresceins; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008239:Lysine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D014227:Triazines
chemical_list: D005452:Fluoresceins; D007457:Iodine Radioisotopes; D011506:Proteins; D014227:Triazines; C019309:cyanuric chloride; D008239:Lysine; D019793:Fluorescein