Reaction of fluorogenic reagents with proteins: II: Capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465

Abstract

The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label α-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 × 10 −4 cm 2 V −1 s −1. The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM α-lactalbumin ( r 2 = 0.99), with a 3 σ detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, α-chymotrypsinogen A, and α-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.