Luminescence properties of pyron red—A new molecular probe for protein research

Abstract

The dependence of the luminescence of the new anionic dye Pyron Red (PR) on the polarity of the medium is investigated. Upon passage from an aqueous phase to a nonpolar phase, PR shows a shortwave shift of the fluorescence emission maximum from 675 to 650 nm and an increase in the fluorescence quantum yield from 0.03 to 0.54–0.70. When complexed with human serum albumin, PR shows fluorescence excitation and emission maxima at 525 and 625 nm and a fluorescence quantum yield of 0.8. In a comparison of the luminescence properties of PR with those of the well-known probes ANS and K35 in water and a complex with albumin, PR is shown to have the maximum absolute sensitivity but a lower fluorescence enhancement upon binding with a protein compared to ANS. A convenient criterion of the probe sensitivity toward binding with a protein that is defined as the ratio of the fluorescence intensities of the protein-bound and the free probe AF=Fb/Ff is proposed. The value of AF(35) for the PR probe ranks between those for the K35 probe with a low AF(18) and ANS with a high AF(105).