A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.
In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent attachment of chromophores to the loop that could subsequently be tested for their ability to sensitize the luminescence of Tb3+ or Eu3+ bound in the loop. 7-Diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin was the most efficient Eu3+ sensitizer studied, consistent with a mechanism of energy transfer that involves the triplet state of the donor. 4-Iodoacetamidosalicylic acid was the most efficient Tb3+ donor tested. Levels of lanthanide ion and labeled C3 as low as 5 x 10(-10) mol/liter could be detected. This protein chelator system has potential to be a useful, flexible complement to the organic chelators currently used in lanthanide-based, time-resolved luminescence immunoassays.
Clark
I D
ID
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
MacManus
J P
JP
Banville
D
D
Szabo
A G
AG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D005063:Europium; D008163:Luminescent Measurements; D008674:Metals, Rare Earth; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D013725:Terbium
D002135:Calcium-Binding Proteins; D008674:Metals, Rare Earth; C034733:oncomodulin; D013725:Terbium; D005063:Europium