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Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye: Noncovalent labeling and nonequilibrium measurement of association constants
Author(s) -
Sloat Amy L.,
Roper Michael G.,
Lin Xiuli,
Ferrance Jerome P.,
Landers James P.,
Colyer Christa L.
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700808
Subject(s) - chemistry , capillary electrophoresis , equilibrium constant , intermolecular force , chromatography , non covalent interactions , analytical chemistry (journal) , molecule , hydrogen bond , organic chemistry
In response to a growing interest in the use of smaller, faster microchip (μ‐chip) methods for the separation of proteins, advancements are proposed that employ the asymmetric squarylium dye Red‐1c as a noncovalent label in μ‐chip CE separations. This work compares on‐column and precolumn labeling methods for the proteins BSA, β‐lactoglobulin B (β‐LB), and α‐lactalbumin (α‐LA). Nonequilibrium CE of equilibrium mixtures (NECEEM) represents an efficient method to determine equilibrium parameters associated with the formation of intermolecular complexes, such as those formed between the dye and proteins in this work, and it allows for the use of weak affinity probes in protein quantitation. In particular, nonequilibrium methods employing both μ‐chip and conventional CE systems were implemented to determine association constants governing the formation of noncovalent complexes of the red luminescent squarylium dye Red‐1c with BSA and β‐LB. By our μ‐chip NECEEM method, the association constants K assoc for β‐LB and BSA complexes with Red‐1c were found to be 3.53×10 3 and 1.65×10 5 M −1 , respectively, whereas association constants found by our conventional CE‐LIF NECEEM method for these same protein–dye systems were some ten times higher. Despite discrepancies between the two methods, both confirmed the preferential interaction of Red‐1c with BSA. In addition, the effect of protein concentration on measured association constant was assessed by conventional CE methods. Although a small decrease in K assoc was observed with the increase in protein concentration, our studies indicate that absolute protein concentration may affect the equilibrium determination less than the relative concentration of protein‐to‐dye.