F. Meadows et al., Determination of protein-dye association by near infrared fluorescence-detected circular dichroism, TALANTA, 50(6), 2000, pp. 1149-1155
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.