Bioconjugate Chem. 2001, 12, 845−849
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ARTICLES A Derivative of Diethylenetriaminepentaacetic Acid for Europium Labeling of Proteins Reiko T. Lee and Yuan C. Lee* Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218. Received September 19, 2000; Revised Manuscript Received April 27, 2001
Preparation of a reagent that will incorporate diethylenetriaminepentaacetic acid (DTPA) into proteins under mild conditions and make a strong europium chelate is described. Aminoacetaldehyde diethyl acetal was reacted with DTPA dianhydride, and mono- and disubstituted products as well as unsubstituted DTPA were separated by gel filtration. The monosubstituted product, after conversion into the corresponding aldehyde by mild acid hydrolysis, is conjugated to protein or other aminocontaining compounds via reductive amination at neutral pH. Although the DTPA-Eu-labeled proteins are themselves not fluorescent, a strong fluorescence of europium can be generated easily by the dissociation-enhancement mechanism. A direct measurement of lectin-ligand interaction using Eulabeled ligand and lectin immobilized on 96-well plate illustrates that the assay utilizing Eu fluorescence is as sensitive as the radioactive assays.
INTRODUCTION
Radioactive labeling has been an indispensable tool in the investigation of biological activities that requires high sensitivity and specificity. In recent years, however, the use of fluorescence of lanthanide chelates (e.g., europium and terbium) has been gaining favor as a viable alternative to the radioactivity, since lanthanide fluorescence has high sensitivity (detection level in the sub-pmol range) and its fluorescence is more specific than that of conventional fluorescent probes (1, 2). The higher specificity and sensitivity result from the fact that the fluorescence emission of these lanthanide ions is narrowbanded with a long Stokes shift and a very long fluorescence decay time (3). The latter two characteristics allow detection of lanthanide fluorescence in the presence of background fluorescence derived from biological samples and common plastic ware, especially if the time-resolved fluorescence measurement is made. The obvious advantage of lanthanide is that there is no need for special handling, including the waste disposal, such as that required for radioactive materials. However, the major disadvantage of lanthanide labeling is the requirement for incorporating a chelating agent, a structure much larger than radioisotopes, which may affect the property of the compound labeled. For this reason it is prudent to aim for minimum level of * Corresponding author: Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218. Tel: 410-516-7041; Fax: 410-516-8716; e-mail:
[email protected]. 1 Abbreviations used: BCA, bicinchoninic acid; BSA, bovine serum albumin; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; TPTZ, 2,4,6-tripyridyl-s-triazine; TFA, trifluoroacetic acid, RCA-I, Ricinus communis agglutinin I.
incorporation of a chelater (ideally
