Abstract
Labeling proteins with fluorescent dyes offers a powerful tool for monitoring protein interactions in vitro and in vivo. In order for this tool to be effective, the nature of the dyes - absorbance and emission properties, solution stability, pH range, and mechanisms for protein interaction - must first be considered. Two new asymmetric, squarylium dyes, bis-SQHN-4d and SQHN-3c, were shown to be only weakly fluorescent in aqueous buffers in the absence of proteins. However, their spectra showed a dramatic increase in fluorescence intensity upon the addition of Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) as model proteins. The enhanced fluorescence properties, attributed to noncovalent binding, allowed the use of the new squarylium dyes as probes for the low-level detection of proteins in a mixture (including myoglobin (pI=7.16), transferrin (pI=5.9), and HSA (pI=4.8)), separated by Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF). Because of the low background fluorescence of these probes, on-column labeling was feasible and led to simple and rapid protein detection. This labeling protocol offered greater sensitivity than the more conventional pre-column labeling protocol (with a 10-fold lower limit of detection for HSA with bis-SQHN-4d). A limit of detection for HSA (by CE-LIF with on-column labeling with bis-SQHN-4d) of 3.42 x 10-8 M indicates that this dye is well suited to the development of other protein assays.
Highlights
The implication of proteins in various biological functions and as markers of various states of disease provides the impetus for developing improved protein assays
Spectral properties of the novel squarylium dyes (7.00 x 10-6 M bisSQHN-4d or 7.50 x 10-6 M SQHN-3c) were determined under various solution conditions in order to assess their suitability as fluorescent probes for proteins
The wavelengths of maximum absorbance and emission for SQHN-3c exceeded those for bis-SQHN-4dThese longer wavelengths can be beneficial because they allow the optical detection to be shifted away from the region of native protein absorbance bands and possible autofluorescence of biological matrices, reducing interference and improving detection limits
Summary
The implication of proteins in various biological functions and as markers of various states of disease provides the impetus for developing improved protein assays. Fluorescence-based methods have been shown to be some of the most sensitive methods for protein analysis [5]. By combining the sensitivity and selectivity of Laser-Induced Fluorescence (LIF) detection with the high efficiency and speed of Capillary Electrophoresis (CE) separations of proteins, many have shown the utility of this system for protein analysis [6,7,8,9,10,11,12,13]. The exact nature of these interactions is often difficult to determine, but evidence of interaction is clearly provided by a change in the emission (wavelength and/or intensity) of the fluorophore–protein complex relative to that of the free, uncomplexed fluorophore, which can be monitored by fluorimetric studies [8]
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