THIOL-REACTIVE, LUMINESCENT EUROPIUM CHELATES - LUMINESCENCE PROBES FOR RESONANCE ENERGY-TRANSFER DISTANCE MEASUREMENTS IN BIOMOLECULES

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using l uminescence resonance energy transfer measurements [P. R. Selvin, T. M . Rana, and J. E. Hearst (1994) J. Am. Chem. Sec. 116, 6029-6030; P.R. Selvin, and J.E. Hearst (1994) Proc. Natl. Acad. Scd. USA 91, 10024-1 0028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds con tain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. Th ese reagents can be used to label proteins at Cys residues or syntheti c oligonucleotides which contain thiol groups. Modification can be rev ersed easily by treatment with a reducing agent (dithiothreitol). Lumi nescence energy transfer between these new chelates and CY5 fluorochro me attached to the opposite ends of 15-bp double-stranded DNA was meas ured to test their usefulness for distance measurements in macromolecu les. The distance measured between the chelate (donor) and CY5 (accept or) was in the range expected for the length of 15-bp DNA. The stabili ty of europium chelates and their conjugates with a protein, the preci sion of distance measurements using these chelates, possible errors du e to intramolecular energy transfer, and the modulation of the Ro valu e with deuterium oxide were tested. The results obtained fully confirm ed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence reson ance energy transfer. (C) 1997 Academic Press.