A phosphorimeter integral to the SPEXFLUOROMAX-P or as an accessory on the SPEX FLUOROLOG system can uncover important information on various systems of chemical and biochemical interest, by revealing data normally masked by intense but rapid fluorescence.
This Application Note describes an example, using the SPEX phosphorimeter, of energy-transfer from a peptide-terbium-complex donor to a fluorescein acceptor. The peptide in the complex absorbs light at 280 nm, the terbium phosphoresces at 485 nm, where fluorescein dye absorbs. The fluorescence of fluorescein can be observed when the sample is excited at 280 nm, using a phosphorimeter accessory.
Samples of peptide-terbium complex were dissolved in aqueous solution, along with fluorescein in some samples. The measurements were taken on a FLUOROLOG-322 spectrofluorometer: fluorescence with a 450-W CW xenon lamp; phosphorescence with a xenon flash lamp; and an R928 photomultiplier operated at 950 V in the photoncounting mode. The SPEX FL-1042 phosphorimeter accessory includes a dual lamp housing (with both CW and pulsed Xe lamps), and all control electronics. A light pulse excites the sample, and variable delays control when the detection window opens, and for how long. Sample excitation was 280 nm, with 100 flashes measured. For luminescence spectra, the integration time was 0.2 s, except as noted. The scans were taken under ambient room conditions.
Results and Discussion
Figure 1 compares luminescence from the peptide- Tb complex with no delay and a 50-s delay following the excitation light pulse. With the delay, the fluorescence at 363 nm from the peptide disappears, isolating the Tb phosphorescence a