Proteins assays are important in both analytical biochemistry and clinical tests. The common quantitative methods, including Biuret, 1 Bradford 2 and Lowry 3 assays, are spectrophotometric or spectrofluorometric tests through the binding of proteins with organic dyes, where color changes should be observed. However, most of these methods suffer from the disadvantages of low sensitivity and selectivity. Therefore, these dyes, whose interactions with proteins fail to display color changes, are limited for the spectrophotometric and spectrofluorometric assays of proteins.Recent studies have focused on resonance light scattering (RLS), since Pasternack et al. 4 showed that it is a powerful technique to detect chromophore aggregation.
5-10We have applied enhanced RLS signals for analytical purposes, having proved that this technique is exceedingly sensitive to determine trace amounts of nucleic acids and proteins. 8,9 Our former reports have shown that the enhanced RLS signals are associated with the size and the charge-couple of the formed dye-protein or dye-DNA complex. [8][9][10] If the size is the main factor responsible for the enhanced RLS signals, the enhanced RLS signals were found to be in proportion to the molecular weight of the proteins.
10The role of the charge-coupled chromophore, however, is much more complicate and it needs further studied. Besides the negative charges on the molecular structures, the pH and ionic strength of the medium that determines the positive charges of protein molecules, depending on the isoelectric points, have a strong effect on the enhanced RLS intensity. In this work, we studied the interaction of proteins with Fast Green FCF (FCF), and tried to elucidate the roles of the charge-couple played in the dye-protein interaction.FCF is an anionic triarylmethane dye (molecular structure in Fig. 1), acting as a fluorophore for the visualization of the acidophilic cell and tissue structures, 11 and for the determination of tamoxifen citrate.12 Herein we report on its interaction with proteins based on RLS measurements where color changes of the dye upon binding to proteins are scarcely observed. It was found that the enhanced RLS intensity of FCF by trace amounts of proteins at 279.0 nm is proportional to the concentration of proteins below 4.0 µg/ml. Although organic dyes, including azo, triphenylmethane and porphryins, have been reported as being used for the determination of proteins of nanogram, the present protein assay using FCF has a much higher sensitivity than that of the reported RLS methods. 9,10,13-24
Experimental
ApparatusThe RLS spectra and intensities were recorded and measured with a Hitachi F-2500 spectrofluorometer (Tokyo, Japan), while the absorption spectra were obtained using a Tianmei 850 spectrophotometer (Hong Kong, China). An S-10A digital pH meter (Xiaoshan Scientific Instruments Plant, Zhejiang, China) was used to measure the pH values of aqueous solutions, and an MVS-1 vortex mixer (Beide Scientific Instrumental Ltd., Beijing, China) was used to blend th...