BINDING-PROPERTIES OF NEAR-IR DYES TO PROTEINS AND SEPARATION OF THE DYE PROTEIN COMPLEXES USING CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION/

The noncovalent binding of the near-infrared (NIR) dyes, DTTCI (cation ic) and IR-125 (anionic), to several model proteins was investigated w ith the use of steady-state and picosecond laser fluorescence measurem ents. In an aqueous berate buffer (pH = 9.2), minimal fluorescence emi ssion from these NIR dyes was observed. When a protein was added to th e solution, enhancements in the fluorescence emission were found for b oth dyes. Time-resolved fluorescence measurements for IR-125 in the pr esence of the protein, beta-casein, indicated a biexponential decay wi th lifetimes of 195 and 682 ps (chi(2) = 1.94). Our data suggest that these dyes distribute themselves between the hydrophobic core of the p rotein and the interstitial aqueous solution. The dye molecules residi ng in the interior of the protein exhibit enhancements in their fluore scence due to a more favorable microenvironment. The binding and enhan ced fluorescence properties allowed the use of these dyes as noncovale nt stains for the low-level detection of proteins separated via capill ary electrophoresis (CE). Detection limits for some model proteins sep arated by CE and stained with these NIR dyes were found to be superior to those obtained by using UV detection in CE.