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

E. Dadachova, L. L. Chappell, M. W. Brechbiel

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

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 III (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 μM, 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.

Original languageEnglish (US)
Pages (from-to)977-982
Number of pages6
JournalNuclear Medicine and Biology
Volume26
Issue number8
DOIs
StatePublished - Nov 1999
Externally publishedYes

Fingerprint

Ligands
Proteins
Acids
Arsenazo III
Color
Temperature

Keywords

  • Antibodies
  • Conjugates
  • Polyazamacrocycles
  • Radioimmunotherapy
  • Radiolabel ing
  • Spectrophotometry

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Medicine
  • Radiology Nuclear Medicine and imaging

Cite this

Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates. / Dadachova, E.; Chappell, L. L.; Brechbiel, M. W.

In: Nuclear Medicine and Biology, Vol. 26, No. 8, 11.1999, p. 977-982.

Research output: Contribution to journalArticle

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N2 - 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 III (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 μM, 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.

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KW - Radioimmunotherapy

KW - Radiolabel ing

KW - Spectrophotometry

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