Determination of protein-dye association by near infrared fluorescence-detected circular dichroism

Near-infrared (NIR) squarylium dye spectral properties were evaluated by absorption, fluorescence, circular dichroism (CD), and fluorescence-detected circular dichroism (FDCD). Substituents of the two NN dyes differed at R₁ and R₂, located symmetrically on the chromophore. The side chains of NN525 are R₁=hexanoic acid, R₂=butyl sulfonate and R₁=R₂=ethyl for NN127. FDCD signals were first confirmed by denaturing BSA with 2–8 M urea showing a diminution of dye FDCD peaks, but no change occurred in spectral properties of the dyes in urea. This indicated that the observed cotton effects occurred by noncovalent interactions with the secondary structure of the protein. The average BSA–dye association constants found by fluorescence, absorbance, and FDCD were 1.27×10⁶ (n=1) and 3.3×10⁶ M⁻¹ (n=1) for NN127 and NN525 respectively. These values were in good agreement when calculated by the three spectroscopic methods validating the use of NIRFDCD for optical parameter calculations. These results are useful to describe NIR squarylium dye labeling of BSA.