Binding Properties of Near-IR Dyes to Proteins and Separation of the Dye/Protein Complexes Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Abstract

The noncovalent binding of the near-infrared (NIR) dyes, DTTCI (cationic) and IR-125 (anionic), to several model proteins was investigated with the use of steady-state and picosecond laser fluorescence measurements. In an aqueous borate buffer (pH = 9.2), minimal fluorescence emission from these NIR dyes was observed. When a protein was added to the solution, enhancements in the fluorescence emission were found for both dyes. Time-resolved fluorescence measurements for IR-125 in the presence of the protein, β-casein, indicated a biexponential decay with lifetimes of 195 and 682 ps (χ² = 1.94). Our data suggest that these dyes distribute themselves between the hydrophobic core of the protein and the interstitial aqueous solution. The dye molecules residing in the interior of the protein exhibit enhancements in their fluorescence due to a more favorable microenvironment. The binding and enhanced fluorescence properties allowed the use of these dyes as noncovalent stains for the low-level detection of proteins separated via capillary electrophoresis (CE). Detection limits for some model proteins separated by CE and stained with these NIR dyes were found to be superior to those obtained by using UV detection in CE.