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

A simple spectrophotometric assay for determination of bifunctional polyaza carboxylate-macrocyclic ligands of different sizes that are conjugated to p roteins has been developed for: 12-membered macrocycle DOTA (2[4 nitrobenzy l]-1, 4, 7, 10-tetraazacyclododecane-N, N',N", N"'-tetraacetic acid) and an alogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]-1,4,7,10,13-penta azacyclopentadecane-N,N',N",N''',N""-pentaacetic acid), and the large 18 me mbered macrocycle HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N",N"',N "",N""'-pentaacetic acid). The method is based on titration of the blue-col ored 1:1 Pb(II)-Arsenazo III (AAIII) complex with the polyazacarboxylate ma crocyclic ligand in the concentration range of 0-2.5 mu M, wherein color ch ange occurring upon transchelation of the Pb(II) from the AAIII to the poly azamacrocyclic ligand is monitored at 656 nm. The assay is performed at amb ient temperature within 20 min without any interfering interaction between the protein and Pb(II)-AA(III) complex. Thus, this method also provides a l igand-to-protein ratio (VP ratio) that reflects the effective number of lig ands per protein molecule available to radiolabeling. The method is not sui table for 14-membered TETA macrocycle 2-[4-nitrobenzyl]-1, 4, 8, 11-tetraaz acyclotetradecane N,N',N",N"'-tetraacetic acid) because of low stability co nstant of Pb(II)-TETA complex. The method is rapid, simple and may be custo mized for other polyazacarboxylate macrocyclic ligands. Published by Elsevi er Science Inc.