Portal de Periódicos da CAPES

Reaction of fluorogenic reagents with proteins

2008; Elsevier BV; Volume: 1194; Issue: 2 Linguagem: Inglês

10.1016/j.chroma.2008.04.047

1873-3778

Kristian E. Swearingen, Jane A. Dickerson, Emily H. Turner, Lauren M. Ramsay, Roza Wojcik, Norman J. Dovic̀hi,

Mass Spectrometry Techniques and Applications

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 cm2 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 (r2 = 0.99), with a 3σ 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.