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

Near-infrared (NIR) squarylium dye spectral properties were evaluated by ab sorption, fluorescence, circular dichroism (CD), and fluorescence-detected circular dichroism (FDCD). Substituents of the two NN dyes differed at R-1 and R-2, located symmetrically on the chromophore. The side chains of NN525 are R-1 = hexanoic acid, R-2 = butyl sulfonate and R-1 = R-2 = ethyl for N N127. FDCD signals were first confirmed by denaturing BSA with 2-8 M urea s howing a diminution of dye FDCD peaks, but no change occurred in spectral p roperties of the dyes in urea. This indicated that the observed cotton effe cts occurred by noncovalent interactions with the secondary structure of th e protein. The average BSA-dye association constants found by fluorescence, absorbance, and FDCD were 1.27 x 10(6) (n = 1) and 3.3 x 10(6) 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 NIRFDC D for optical parameter calculations. These results are useful to describe NIR squarylium dye labeling of BSA. (C) 2000 Elsevier Science B.V. All righ ts reserved.