Comparison of Three Common Amine Reactive Fluorescent Probes Used for Conjugation to Biomolecules by Capillary Zone Electrophoresis

The conjugation of three amine reactive fluorescent probes, each containing the fluorophore fluorescein but different reactive moieties, was compared using the protein myoglobin and the amino acid L-lysine as reagents. The three different reactive moieties were an isothiocyanate group (FITC), a succinimidyl ester group (CFSE), and a dichlorotriazine group (DTAF). The relative performance was based on the degree of conjugation to myoglobin, the rate of reaction, freedom from hydrolysis, and the stability of a conjugate with lysine. Performance was evaluated by separating the conjugation reaction reagents and products on-line, using capillary zone electrophoresis, and assessing relative amounts by absorbance detection. Each of the reactive probes demonstrated the ability to achieve a similar degree of conjugation, which depended mostly on allowed conjugated reaction time, and a rate of conjugation that rendered hydrolysis of the reactive moiety insignificant. For the relative rate of conjugation between probes and stability of the resulting conjugate, CFSE demonstrated superior performance, followed by DTAF and then FITC, for both the protein myoglobin and the amino acid L-lysine. The FITC conjugation reaction was much easier to control, however, which may be significant for applications that require a precise degree of conjugation. With regard to conjugate-bond stability, the FITC conjugate demonstrated inferior performance when subjected to incubation at 37 degrees C.