COMPARISON OF 3 COMMON AMINE REACTIVE FLUORESCENT-PROBES USED FOR CONJUGATION TO BIOMOLECULES BY CAPILLARY ZONE ELECTROPHORESIS

The conjugation of three amine reactive fluorescent probes, each conta ining the fluorophore fluorescein but different reactive moieties, was compared using the protein myoglobin and the amino acid L-lysine as r eagents. The three different reactive moieties were an isothiocyanate group (FITC), a succinimidyl ester group (CFSE), and a dichlorotriazin e group (DTAF). The relative performance was based on the degree of co njugation to myoglobin, the rate of reaction, freedom from hydrolysis, and the stability of a conjugate with lysine. Performance was evaluat ed by separating the conjugation reaction reagents and products on-lin e, using capillary zone electrophoresis, and assessing relative amount s by absorbance detection. Each of the reactive probes demonstrated th e ability to achieve a similar degree of conjugation, which depended m ostly on allowed conjugated reaction time, and a rate of conjugation t hat rendered hydrolysis of the reactive moiety insignificant. For the relative rate of conjugation between probes and stability of the resul ting conjugate, CFSE demonstrated superior performance, followed by DT AF and then FITC, for both the protein myoglobin and the amino acid L- lysine. The FITC conjugation reaction was much easier to control, howe ver, which may be significant for applications that require a precise degree of conjugation. With regard to conjugate-bond stability, the FI TC conjugate demonstrated inferior performance when subjected to incub ation at 37 degrees C.