Send to

Choose Destination
Nucl Med Biol. 1999 Nov;26(8):977-82.

Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates.

Author information

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.


A simple spectrophotometric assay for determination of bifunctional polyazacarboxylate-macrocyclic ligands of different sizes that are conjugated to proteins has been developed for: 12-membered macrocycle DOTA (2-[4-nitrobenzyl]-1, 4, 7, 10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and analogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N ''',N''''-pentaacetic acid), and the large 18-membered macrocycle HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''''- hexaacetic acid). The method is based on titration of the blue-colored 1:1 Pb(II)-Arsenazo II (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 microM, wherein color change occurring upon transchelation of the Pb(II) from the AAIII to the polyazamacrocyclic ligand is monitored at 656 nm. The assay is performed at ambient temperature within 20 min without any interfering interaction between the protein and Pb(II)-AA(III) complex. Thus, this method also provides a ligand-to-protein ratio (L/P ratio) that reflects the effective number of ligands per protein molecule available to radiolabeling. The method is not suitable for 14-membered TETA macrocycle (2-[4-nitrobenzyl]-1, 4, 8, 11-tetraazacyclotetradecane N,N',N'',N'''-tetraacetic acid) because of low stability constant of Pb(II)-TETA complex. The method is rapid, simple and may be customized for other polyazacarboxylate macrocyclic ligands.

[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center